DEVELOPMENT OF NEW DRUGS FOR TB CHEMOTHERAPY

Analysis of the current drug pipeline1Table of contents
TABLE OF CONTENTS ................................................................................................................. 2
ACKNOWLEDGEMENTS............................................................................................................... 4
SUMMARY ..................................................................................................................................... 5
1. INTRODUCTION ........................................................................................................................ 8
2. TARGETS AND MODE OF ACTION OF CURRENT TB DRUGS............................................... 8
3. WHY NEW DRUGS ARE NEEDED? .......................................................................................... 9
4. THE NEW TB DRUG PIPELINE ............................................................................................... 12
4.1 Novel chemical entities.................................................................................................... 13
Diarylquinoline TMC207 (Johnson & Johnson) .................................................................. 13
Nitroimidazole PA-824 (Chiron Corp.-TB Alliance) ............................................................ 14
Nitroimidazole OPC-67683 (Otsuka Pharmaceuticals, Japan) ........................................... 15
Pyrrole LL- 3858 (Lupin Limited, India) .............................................................................. 16
Pleuromutilins (GlaxoSmithKline-TB Alliance Partnership) ................................................ 16
Dipiperidine SQ-609 (Sequella Inc.) .................................................................................. 16
ATP Synthase Inhibitor FAS20013 (FASgene) .................................................................. 17
Translocase I Inhibitor (Sequella Inc.) ............................................................................... 17
InhA Inhibitors (GlaxoSmithKline-TB Alliance) ................................................................... 17
Isocitrate Lyase Inhibitors (GlaxoSmithKline-TB Alliance) ................................................. 17
4.2 Compounds originating from existing families of drugs .................................................... 18
Using existing Fluoroquinolones for TB ? .......................................................................... 18
New Quinolones ................................................................................................................ 22
Non-fluorinated quinolones................................................................................................ 23
Diamine SQ-109................................................................................................................ 23
Macrolides......................................................................................................................... 23
Thiolactomycin analogs ..................................................................................................... 23
Nitrofuranylamides ............................................................................................................ 24
Nitroimidazole Analogs ...................................................................................................... 24
4.3 Summary of the drug pipeline.......................................................................................... 25
5. EXPECTED TIMELINES TOWARDS APPROVAL FOR NEW CANDIDATE DRUGS .............. 27
6. A CRUCIAL GAP: LACK OF EARLY STAGE DRUG DISCOVERY ........................................ 29
7. DISCUSSION AND CONCLUSIONS ........................................................................................ 32
Pressing needs still remain.................................................................................................... 32
Time to sow new seeds now as all the low-hanging fruit have been eaten ............................ 36
APPENDIX A: PROMISES FROM THE BASIC RESEARCH FIELD ............................................ 38
Genes involved in energy metabolism and response to oxygen limitation ............................. 39
Genes encoding enzymes of the glyoxylate shunt ................................................................. 40
Genes involved in the response to nutrients limitation ........................................................... 41
Genes involved in cell wall and membrane metabolism......................................................... 42
Genes involved in transcriptional regulation. ......................................................................... 43
Genes involved in promoting M. tuberculosis survival inside macrophages. .......................... 44
Inhibition of phagosome maturation................................................................................... 44
Resistance to nitric oxide stress ........................................................................................ 45
APPENDIX B: UPDATE ON COMPOUNDS IN THE PIPELINE ................................................... 46
Malate Synthase Inhibitors (GSK, Rockefeller University, Texas A&M) ............................. 46
Riminophenazines (Institutes of Materia Medica/BRTI) ..................................................... 46
Capuramycins (Sankyo/Sequella)...................................................................................... 46
2

Proteasome Inhibitors (Cornell University)......................................................................... 47
Protease Inhibitors (Medivir).............................................................................................. 47
Bifunctional Molecules (Cumbre- TB Alliance) ................................................................... 47
Bacterial Topoisomerase Inhibitors (GlaxoSmithKline-TB Alliance) ................................... 48
APPENDIX C: EXTENSIVE DRUG RESISTANT TUBERCULOSIS (XDR-TB) ............................ 49
REFERENCES ............................................................................................................................. 51

Author:Martina Casenghi, PhD
October 20063

Acknowledgements
This report has been written in collaboration with Tido von Schoen-Angerer. We thank Nathan
Ford for useful comments on the manuscript and for his substantial contribution to the Chapter 7 ofthis report. We also thank Maria Freire, Mel Spigelman, Ann Ginsberg, Zhenkun Ma and Nina
Schwalbe (TB Alliance) for giving valuable contribution and sharing useful information. Special
thanks are due to Carl Nathan (Weill Medical College, Cornell University, New York), Stefan
Kaufmann (Max-Planck Institute for Infection Biology, Berlin), David Sherman (University ofWashington, Seattle), Cristophe Guilhot (Institute de Pharmacologie Structurale, CNRS, Toulouse),
Valerie Mizrahi (University of the Witwatersrand, Johannesburg), John McKinney (Rockefeller
University, New York), Ken Duncan (Imperial College London), David Kusner (University of Iowa
Carver College of Medicine, Iowa City) for helpful contributions and stimulating discussions. We
are grateful to Francesca Antonelli, Ravindra Babu Chalmalasetty, Arianna Casciati and Catherine
Regnard for their help in collecting scientific literature and to Guido Panté for support and helpful
discussions.

4Summary
With approximately 9 million people developing active tuberculosis (TB) every year and 1.7
million deaths annually, TB is far from under control. Human immunodeficiency virus (HIV)infection dramatically increases the risk of developing active tuberculosis and is driving the TB
epidemic in Africa. HIV renders tuberculosis more difficult to diagnose (due to higher incidence of
sputum negative disease), and treat (due to interactions and side-effects). The increasing spreadof multidrug-resistant TB (MDR-TB) and the recalcitrant nature of persistent infections pose
additional challenges to treatment with currently available anti-TB drugs. The situation isexacerbated by the increasing emergence of extensively drug-resistant (XDR) TB. Resistance to atleast two main first-line drugs and additionally to three or more of the six classes of second-line
drugs makes this form of TB virtually untreatable with available drugs.
Although TB can be cured, current treatment is complex and long lasting, involving four drugs
for 2 months and two drugs for at least another 4 months. Directly Observed Therapy (DOT), as
promoted by the World Health Organisation (WHO) to improve compliance for the difficult andlong-lasting regimen, is demanding for patients, labour intensive for health staff and iscompromised in settings where health services are poorly accessible. MDR-TB is even more
complex and expensive to treat, and in developing countries treatment is limited to a few projects
with limited numbers of patients.
After decades of standstill in TB drug development, the drug pipeline has begun to fill up during
the last 5 years. Established in 2000 and largely funded by the Bill & Melinda Gates Foundation,
the Global Alliance for TB Drug Development (TB Alliance) has played a critical role in changing
the TB research and development (R&D) landscape and is associated with approximately half of allcompounds (or projects aimed to identify candidate compounds) in development. The main criteria
established by the TB Alliance to select drug candidates for further development are shortening of
the current treatment, activity against MDR-TB and lack of interactions with antiretroviral drugs
represent.
During the last years, increased public awareness of the lack of R&D for neglected diseases
has led at least one pharmaceutical company to establish an institute undertaking R&D activities intuberculosis on a ‘no-profit-no-loss’ basis. Other companies have engaged in tuberculosis R&D on
for-profit basis, and with some success: three of the six anti-TB candidate drugs currently in clinical
trials have been developed by for-profit companies.
Major advances have been also made in basic research. Modern molecular and genetic tools
have become available for Mycobacterium tuberculosis (such as targeted mutagenesis, arraybased analysis of mutant libraries, techniques for conditional gene silencing, and global gene
expression profiling) and this has led to impressive improvements in the knowledge andunderstanding of the basic biology and physiology of M. tuberculosis. These progresses were5

largely supported by major funding programmes from NIH/NIAID, the Wellcome Trust, and the EU
during the 1990s.
Despite these positive changes there are still problems that need to be tackled. A criticalquestion today is whether they are sufficient to bring improved treatment to patients in the next few
years.
A first challenge concerns the sustainability of the current effort. As promising compounds moveinto expensive clinical trials, PDPs such as the TB Alliance face a significant funding gap. These
initiatives are driven almost entirely by philanthropic effort, with governments only contributing ameagre 16% to PDPs engagement in drug development. Financial support will need to increase toensure that the development of these promising new compounds is supported all the way to trials.
The next important question is whether there are a sufficient number of promising compounds in
the TB pipeline for a broadly effective new treatment combination to be developed. Althoughdifferent attrition rates might apply, the number of candidate compounds is still small compared tothe drug pipelines for diseases of major concern to wealthy countries such as cancer or
cardiovascular diseases (and the number of companies engaged in the latter is also greater).
Furthermore, many of the compounds in the pipeline are either derivatives of existing
compounds or they target the same cellular processes as drugs currently in use. Whilst analogues
and derivatives are far quicker to develop, they may be subject to cross-resistance, as has been
the case with the new rifamycins and quinolones.
Modern technologies and rational approaches to drug design (such as creation of genomic
libraries of M. tuberculosis conditional knock-out mutants for comprehensive target identificationand validation, target-based drug discovery, or determination of three-dimensional crystal structureof molecular targets) are still weakly implemented in the field of drug discovery for tuberculosis.
Even the more promising candidate compounds currently in clinical development were identified
serendipitously in screenings that were not designed originally for activity against M. tuberculosis.
There is consensus among the TB scientific community that in order to obtain a real breakthroughin TB therapy and drastically shorten treatment there is an urgent need for rational approaches
aimed at tackling the problem of mycobacterial persistence. The adaptations that allow M.
tuberculosis to persist in the host despite a vigorous adaptive immune response likely contribute to
the difficulty in curing TB with current chemotherapy . Although drugs currently in the pipeline could
significantly shorten treatment, it is likely to remain a matter of months rather than weeks or days.
There are two major roadblocks that hamper the implementation of rational drug design in TB
drug discovery. The first is the lack of a comprehensive characterization of the fundamental biology
of mycobacteria as they persist in human tissues, which prevents the identification and validationof potential targets that are relevant for the survival of the bacteria in vivo. The second is the weakengagement into early-stage drug discovery; as a consequence the advanced knowledge about M.

6

tuberculosis metabolism, physiology and genetics is not being translated into validated targets thatcan be used for screening of new lead compounds.
As part of the Grand Challenges in Global Health initiative the Gates Foundation is funding
research into the molecular pathways of persistence, with the aim of novel target identification. In
addition, the Gates Foundation recently announced a new initiative that specifically aims at
accelerating drug discovery for tuberculosis. While acknowledging this significant contribution, it isimportant not to rely exclusively on a single initiative to address a complex scientific problem ofsuch great importance. Much attention must be paid to these critical issues.
If faster progress is to be achieved in drug discovery for tuberculosis then the advanced
knowledge about M. tuberculosis metabolism and physiology needs to be translated into validated
targets that can be used for screening of new lead compounds. A key difficulty lies in securing
sustained funding for translational research projects such as target validation and chemical
genetics. Up to now the major funding bodies for basic research in TB – NIH/NIAID, the Wellcome
Trust, and the EU – give priority to “blue sky” basic research projects and “hypothesis-driven”
science. Rare exceptions are made for occasional grants based on request for application, but
generally it is very difficult for academic labs to obtain funds for projects that fall between the areasof basic and applied research. The private sector for its part is reluctant to engage in early stage
drug discovery projects; drug development is instead only embarked upon when rigorously
validated targets are available or a lead compound has been already identified.
Real improvements in TB treatment will require substantial strengthening of early-stage
discovery research to identify new compounds and targets. Without a thriving background of
discovery-oriented translational research, which is largely dependent on public funding, drug
development is destined to fail in terms of long-term goals for effective TB management. Existing
modern technologies need to be urgently and more comprehensively applied to TB if the pipeline
for drug R&D is to be filled. The reluctance of the pharmaceutical sector to invest in early-stage
discovery research for neglected diseases exacerbates the pressing need to translate basic
scientific knowledge into novel targets and fresh approaches towards improved therapies. Without
proper public engagement in early stage drug discovery and implementation of rational approaches,
progress in innovation will be severely hindered.

7

1. Introduction
Mycobacterium tuberculosis infects about 32% of the world’s population. Every year,
approximately 8 million of these infected people develop active tuberculosis (TB) and almost 2
million of these will die from the disease (WHO, 2005).
Despite 40 years of anti-TB chemotherapy, tuberculosis remains one of the leading infectious
diseases worldwide. Among the main obstacles to the global control of the disease are the HIV
epidemic that has dramatically increased risk for developing active TB, the increasing emergenceof multi-drug resistant TB (MDR-TB) and the recalcitrance of persistent infections to treatment withconventional anti-TB drugs (Corbett et al., 2003; Gomez and McKinney, 2004; Smith et al., 2003).
The situation is exacerbated by the increasing emergence of extensively drug-resistant (XDR) TB(CDC, 2006) . XDR-TB is characterized by resistance to at least the two first-line drugs rifampicin
and isoniazid and additionally to a fluoroquinolone and an injectable drug (kanamycin, amikacin orcapreomycin) among the second-line drugs (WHO Meeting of the Global XDR-TB Task Force).
The extensive resistance makes this form of TB particularly cumbersome to treat with available
drugs (ref). The current situation clearly demonstrates the need for a re-evaluation of our approach
to treating TB. Drug development for tuberculosis and other neglected diseases has been at a
virtual standstill for decades, but increased awareness and advocacy in recent years have led to
new initiatives in TB drug development. Today, the TB drug pipeline is no longer empty as it was
five years ago.
This report provides a detailed analysis of today’s TB drug pipeline in an attempt to see whether
current approaches are likely to provide truly effective new tools to treat tuberculosis.

2. Targets and mode of action of current TB drugs
Current chemotherapy for TB largely relies on drugs that inhibit bacterial metabolism with aheavy emphasis on inhibitors of the cell wall synthesis (for review see Zhang, 2005). According totheir mode of action, first and second line TB drugs can be grouped as cell wall inhibitors (isonizide,
ethambutol, ethionammide, cycloserine), nucleic acid synthesis inhibitors (rifampicin, quinolones),
protein synthesis inhibitors (streptomycin, kanamycin) and inhibitors of membrane energy
metabolism (pyrazinamide). Targets and mechanisms of action of current TB drugs are
summarized in Table 1.
Existing TB drugs are therefore only able to target actively growing bacteria through the
inhibition of cell processes such as cell wall biogenesis and DNA replication. This implies thatcurrent TB chemotherapy is characterized by an efficient bactericidal activity but an extremely
weak sterilizing activity, defined as the ability to kill the slowly growing or slowly metabolisingbacteria that persist after the growing bacteria have been killed by bactericidal drugs (i.e.
8persisters; (Mitchison, 1980). Sterilizing activity also describes the ability to eliminate latent or“dormant” bacteria that survive inside the host macrophages. This bias is hardly surprising as antiTB drugs have traditionally been identified by their ability to suppress or kill replicating cultures ofbacteria in vitro.
The weak sterilizing property of available TB drugs is one of the major drawbacks for current
TB chemotherapy. Although rifampicin (RIF) and pyrazinamide (PZA) are partially sterilizing drugs
and play an important role in shortening the therapy from 12-18 months to 6 months, there are still
populations of persisting bacteria that are not killed by RIF and PZA. Thus, although achieving aclinical cure, the current TB chemotherapy does not achieve a bacteriological cure since thetherapy cannot completely eradicate all bacilli in the lesions (McCune and Tompsett, 1956).
Table 1. Commonly used TB drugs and their targets (adapted from Zhang, 2005)

3. Why new drugs are needed?
HIV has dramatically increased the risk of developing active tuberculosis and HIV co-infection
makes tuberculosis more difficult to diagnose (due to more complicated presentations) and treat(due to interactions and side-effects). The increasing emergence of multi-drug resistant TB (MDR-

9

TB) and the recalcitrant nature of persistent infections pose additional challenges to treatment withconventional anti-TB drugs.
Although TB can be cured with current drugs treatment is complex and long-lasting, involving
four drugs for two months and two drugs for at least another 4 months. This makes compliancedifficult. Directly Observed Treatment (DOT) as promoted by the World Health Organisation (WHO)to improve compliance for the difficult and long regimen can improve cure rates but is demanding
for patients and labour intensive for health staff (O'Brien and Nunn, 2001).
Why is the treatment for TB with conventional drugs so slow and difficult? In pioneering studies
McDermott and colleagues showed that the efficacy of drugs against M. tuberculosis in vitro was
not matched by their efficiency in vivo (McCune et al., 1956). The difference is striking;exponentially growing cultures of M. tuberculosis can be sterilized in a few days using frontline
bactericidal drugs such as isoniazid and rifampicin, yet the same drug combination requires
months to achieve similar effects against bacteria living in host tissues. Why? The most obvious
explanation would be failure of drugs to achieve optimal levels within TB lesions, but there isevidence that drugs availability is not a limiting factor (Barclay et al., 1953; Clark, 1985). It has
been proposed that persistence of tubercle bacilli in the face of chemotherapy might be attributableto physiologic heterogeneity of bacteria in the tissues (Mitchison, 1979). This idea was inspired andsupported by the long-established observation that slow- and non-growing bacteria arephenotypically resistant or tolerant to killing by antimicrobials (Handwerger and Tomasz, 1985).
According to Mitchison, tubercle bacilli in lesions consist of at least four different populations (seefigure 1):1) Bacteria that are actively growing, killed primarily by isoniazid (INH)
2) Bacteria that have spurts of metabolism, killed by rifampicin (RIF)
3) Bacteria that are characterized by low metabolic activity and reside in acid pH environment,
killed by pyrazinamide (PZA)
4) Bacteria that are “dormant” or “persisters”, not killed by any current TB drug.
During the initial phase of chemotherapy, which lasts for about 2 days, bacilli are killed
exponentially at a rapid rate, followed by a further lengthy period of much slower exponential killing(Jindani et al., 2003). It is assumed that those bacilli killed in the first 2 days are actively multiplying,
while those in the succeeding period are persisters killed by the slower sterilizing activities of thedrugs. As mentioned in the previous section, drugs in the current regimen differ in their relative
bactericidal activities, with the activity of isoniazid predominating during the initial phase, and intheir subsequent sterilizing activity, with the activity of rifampicin and pyrazinamide predominating
during the continuation phase (Dickinson and Mitchison, 1981; Jindani et al., 2003). In an in vitro
model of drug action, a 30-day static culture has been extensively used for the last 60 years andhas been taken to resemble the persister population in its response to drugs (Herbert et al., 1996;
Mitchison, 1992; Mitchison and Selkon, 1956). The drugs added to this static culture have the10same slow sterilizing action that is responsible for the prolongation of therapy. Thus, evidence
suggests that activity against the population of persistent bacilli ultimately determines the durationof therapy necessary for a given regimen to sterilize lesions and provide a stable cure of the host(Grosset and Ji, 1998). From this there is evidently an urgent need to develop new and more
effective TB drugs that are not only active against MDR-TB but also shorten the length of treatment
targeting non-replicating persistent bacilli.

Figure 1. TB drugs targeting distinct M. tuberculosis subpopulation (adapted from Zhang, 2005)

As emphasized by the Guidelines for Tuberculosis drug development issued by the Global
Alliance for TB Drug Development (TB Alliance) (Global Alliance for TB drug development, (2001);
http://www.tballiance.org/pdf/TB%20Scientific%20Blueprint%20Full.pdf)

a

new

TB

treatment

should offer at least one of the following three improvements over the existing regimens:•

shorten the total duration of treatment and/or significantly reduce the number of doses needed
to be taken under DOT•

improve the treatment of MDR-TB•

provide a more effective treatment of latent TB infection

Shortening of the current treatment, activity against MDR-TB, and lack of liver enzyme induction
and inhibition (to avoid interactions with antiretrovirals) are the main criteria the TB Alliance isusing to select drug candidate that should be pursued for further development. Finding a treatment

11

for latent TB is currently not a strategic priority for the TB Alliance as it considers treatment ofactive TB as a more feasible achievement to be reached in a short-term perspective.
In order to shorten the development time for a new regimen, TB Alliance is working on both
identifying individual novel compounds and developing new drug combinations. TB Alliance iscurrently engaged in discussions with regulatory authorities (FDA and EMEA) to see how they cantest new compounds simultaneously rather than consecutively. Indeed, the conventional approachto drug development requires to substitute each drug in the current regimen singularly, only after
each new drug has been approved. Considering that it takes on average 6 years for a new drug tobe registered, the development of a completely new first line regimen could take approximately 24
years. TB Alliance’s innovative proposal of testing new compounds simultaneously could
drastically shorten the procedure, but ethical implications have to be taken in strong consideration
in identifying a practical way to implement such clinical trial design.

4. The new TB drug pipeline
Drug development for tuberculosis and other neglected diseases has been at a standstill for
decades. Today however, thanks also to the work of the Global Alliance for TB Drug Development(TB Alliance), the TB drug pipeline is richer than it has been in the last forty years. Created in
20001 and largely funded by the Bill & Melinda Gates Foundation, the TB Alliance is a productdevelopment partnership (PDP) that focuses on both pre-clinical and clinical development ofcandidate compounds for TB chemotherapy. The TB Alliance is associated with approximately halfof all compounds (or projects aimed to identify candidate compounds) currently being developed.
In addition to this, increased public awareness on the lack of R&D for neglected diseases inrecent years have led some multinational pharmaceutical companies to set up Research andDevelopment (R&D) institutes or initiatives in drug development for tuberculosis, dengue, malaria
and leishmaniasis on a ‘no-profit-no-loss’ basis. Among the multinational pharmaceuticalcompanies currently involved in anti-TB drug R&D are Novartis, AstraZeneca and GlaxoSmithKline(GSK). Smaller pharmaceutical companies have also engaged in neglected disease R&D on acommercial basis (Moran et al., 2005), and with some success: two of the anti-TB candidate drugs
currently in clinical trials have been developed by medium-size pharmaceuticals companies suchas Lupin Limited (India) and Otsuka Pharmaceuticals (Japan).

The global TB drug pipeline as reported by the Stop TB partnership working group on new TB
drugs(http://www.stoptb.org/wg/new_drugs/assets/documents/WGND%20Strategic%20Plan%20(final).p1

MSF took an active role in the founding of the TB Alliance and former MSF president James Orbinski became thefirst president of the TB Alliance stakeholder Association right after he left MSF

12df) is summarised in Table 2. This is an overview of all drug candidates in the pipeline, belonging
to different entities and not only the TB Alliance. In order to analyse the pipeline it is useful to groupdrug candidates currently in two main categories:
1) Novel chemical entities
2) Compounds originating from existing families of drugs, where innovative chemistry is used
to optimise the compounds
Table 2. Global TB drug pipeline March 2006 (kindly provided by Stop TB Partnership working groupon new TB drugs)

4.1 Novel chemical entities
Diarylquinoline TMC207 (Johnson & Johnson)
Diarylquinoline TMC207 is an extremely promising member of a new class of anti-mycobacterial
agents. To date, 20 molecules of the diarylquinoline series have been shown to have a minimum
inhibitory concentration below 0.5 μg/ml against M. tuberculosis H37Rv. Antimicrobial activity wasconfirmed in vivo for three of these compounds (Andries et al., 2005). The most active compoundof the class is TMC207 and its spectrum is unique in its specificity to mycobacteria. The target andmechanism of action of diarylquinoline TMC207 is different from those of other anti-TB agents
implying low probability of cross-resistance with existing-TB drugs. This is further suggested by thefact that diarylquinoline TMC207 is able to inhibit bacterial growth when tested on MDR-TB isolates.
Diarylquinoline TMC207 seems to act by inhibiting the ATP synthase ( Andries at al., 2005;Petrella
et al., 2006), leading to ATP depletion and pH imbalance. This new anti-mycobacterial compound13

has potent early bactericidal activity in the non-established infection murine mouse model,
matching or exceeding that of isoniazid. Moreover, diarylquinoline TMC207 has potent latebactericidal activity in the established infection in murine TB model. Substitution of rifampicin,
isoniazid or pyrazinamide with diarylquinoline TMC207 accelerated activity leading to complete
culture conversion after 2 months of treatment in some combinations. In particular, thediarylquinoline-isoniazid-pyrazinamide and diarylquinoline-rifampicin-pyrazinamide combinations
cleared the lungs of TB in all the mice after two months. Diarylquinoline TMC207 has been also
tested in various combination with the second line drugs amikacin, pyrazinamide, moxifloxacin andethionamide in mice infected with the drug-susceptible virulent M. tuberculosis strain H37Rv.
Diarylquinoline containing regimen were more were more active than the current recommended
regimen for MDR-TB amikacin-pyrazinamide-moxifloxacin-ethionamide and culture negativity ofthe both lungs and spleens was reached after 2 months of treatment in almost every case(Lounis
et al., 2006). A thorough assessment of dyarilquinoline activity against MDR-TB in vivo would
however require testing of animal models infected with multi-drug resistant bacterial strains rather
than with drug-susceptible strains.
Pharmacokinetic and pharmacodynamic studies in mice showed long plasma half-life, hightissue penetration and long tissue half-life. These are all attributes that are valuable for treatmentof chronic infections and may also be important for development of simpler dosing regimens(Andries et al., 2005).
Originally identified by Johnson&Johnson scientists diarylquinoline TMC207 has been transferredto Tibotec Pharmaceuticals Limited (a J&J subsidiary company) for further clinical development
and it is now refered to as TMC207. Preliminary studies in mouse models indicate thatdiarylquinoline TMC207 has sterilizing activity in vivo. Studies in mice also showed potential
reduction of treatment duration. Diarylquinoline TMC207 is currently in phase IIa clinical trials(Tibotec/Johnson & Johnson personal communication)

Nitroimidazole PA-824 (Chiron Corp.-TB Alliance)
Nitroimidazole PA-824 is a new nitroimidazole derivative developed by PathoGenesis-Chiron
and currently being developed by the TB Alliance. The TB Alliance received worldwide exclusive
rights to PA-824 and its analogs for the treatment of TB and Chiron pledged to sell it royalty-free
for endemic countries. After activation by a mechanism dependent on M. tuberculosis F420 factor,
PA-824 acts mainly by inhibiting the synthesis of cell wall components through molecular targets
that are yet to be identified. In vitro, PA-824 showed high activity against drug-sensitive and drugresistant M. tuberculosis strains, indicating that there is no cross-resistance with current TB drugs.
Moreover, PA-824 exhibited bactericidal activity against both replicating and static bacteria in vitro(Stover et al., 2000). PA-824 bactericidal activity against non-replicating bacteria was comparable
to that of RIF (Lenaerts et al., 2005). Experiments performed in mice showed that administration of14

PA-824 at doses ranging from 25 to 100 mg/ml produced reductions in the bacterial burden in
spleen and lungs that were comparable to that produced by INH at 25 mg/ml (Stover et al., 2000;
Tyagi et al., 2005). In order to test for possible sterilizing activity the compound was tested incontinuation phase in mouse models that had received RHZ for 2 months. Although PA-824 wassignificantly more efficient than isoniazid or moxifloxacin in clearing the infection during thecontinuation phase, it was not better than that of rifampicin+isoniazid combination (Tyagi et al.,2005). In long-term treatment experiments performed to determine its sterilizing capacity,administration of PA-824 as monotherapy in mice led to a decrease in bacterial counts in the lungs
comparable to that obtained with rifampicin or isoniazid monotherapy. After 12 weeks of treatment
with PA-824, rifampicin or isoniazid, a complete eradication of the bacterial load was not achievedin any of the treated mice (Lenaerts et al., 2005). When a 6-month treatment regimen containing
PA-824 in combination with rifampicin, isoniazid and pyrazinmide was tested in mice, any of thePA-824 containing regimens resulted superior to the standard first line regimen in terms of more
rapid reductions of the bacterila burden during treatment and lower rates of relapse after treatment(Nuemberger et al., 2006). Further investigations are required to assess the potentiality of PA-824
to improve the treatment of both drug-susceptible and multi-drug resistant tuberculosis when usedin novel combinations with new drug candidates in addition to existing antituberculosis drugs. PA824 entered phase I clinical trials in June 2005.

Nitroimidazole OPC-67683 (Otsuka Pharmaceuticals, Japan)
Little information about this compound is publicly available. It belongs to a subclass of mycolicacid inhibitors, thus it interferes with the biosynthesis of the mycobacteria cell wall. Minimum
inhibitory concentrations (MICs) of this compound were determined using standard and clinicallyisolated M. tuberculosis strains, including multi-drug resistant strains. In vitro, OPC-67683 showed
high activity against drug-sensitive as well drug-resistant strains with MICs ranging 6 - 24 ng/mL.
No cross-resistance with any of the current first-line drugs was observed. Moreover, OPC-67683
showed strong intracellular activity against H37Rv strain of M.tuberculosis residing within humanmacrophages and type II pneumocytes. Studies in animal models showed that OPC-67683 iseffective against sensitive (H37v) and MDR-TB strains in vivo starting from a concentration of
0.03125 mg/body. Furthermore, OPC showed in vivo efficacy against H37Rv strain even in SCID
mice (affected by a severe immune deficiency and used as a model for AIDS), starting form aconcentration of 0.00781 mg/body. When tested in mouse models for chronic tuberculosis, OPC67683 showed a 6-7 fold higher activity compared to first-line drugs isoniazid and rifampicin. No
antagonist activity could be observed when OPC-67683 was used in combination with currently
used anti-TB drugs in vivo. Pharmacokinetics studies in mice, rats and dogs revealed that thiscompound is relatively well absorbed after oral dosing at 3 mg/kg. The bioavailability in eachspecies was 35-60% with a concentration 3-7 times higher in the lung than in the plasma. The

15

compound was well distributed in most tissues (Abstracts submitted to 45th Interscience
Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Washinghton DC Dec 1619/2005

http://www.icaac.org/).

The

TB

Allianceiscurrently

negotiating

with

OtsukaPharmaceuticals concerning the further joint development of this compound and the project is in
discussion.

Pyrrole LL- 3858 (Lupin Limited, India)
Very limited information on the development of pyrroles as anti-mycobacterial agents is currently
available. Pyrroles derivatives were found to be active against standard and drug-sensitive M.
tuberculosis strains in vitro (Deidda et al., 1998; Ragno et al., 2000) Lupin Limited reported theidentification of a Pyrrole derivative (LL-3858) that showed higher bactericidal activity than
Isoniazid when administered as monotherapy to infected mice. In mouse models, a 12 weeks
treatment with LL-3858 plus isoniazid and rifampicin, or LL-3858 plus isoniazid-rifampicinpyrazinamide, sterilized the lungs of all infected mice. Experiments conducted in mice and dogs
showed that the compound is well absorbed, with levels in serum above the MIC and better half-lifeand Cmax than those showed by isoniazid. No information is available concerning the molecularmechanisms that mediate LL-3858’s bactericidal activity (Abstract n.63 submitted to the American
ChemicalSociety

Meeting,AnheimCA,

March

28-April

01

2004;

http://wiz2.pharm.wayne.edu/mediabstracts2004.pdf). Pyrrole LL3858 is currently in Phase I
Clinical Trials (Lupin Limited, personal communication; Dr Federico Gomez de las Heras, GSK at
TB Alliance stakeholder meeting, Paris 2005)

Pleuromutilins (GlaxoSmithKline-TB Alliance Partnership)
The pleuromutilins represent a novel class of antibiotics derived from a natural product. Theyinterfere with protein synthesis by binding to the 23S rRNA and therefore inhibiting the peptide
bond formation (Schlunzen et al., 2004). Despite the novelty of this class of compounds, recent
studies have shown that cross-resistance might occur among pleromutilins and oxazolidinones(Long et al., 2006). Pleuromutilins have been shown to inhibit the growth of M. tuberculosis in vitro.
The goal of this project, launched in collaboration with GSK, is the identification of a pleuromutilinderivative that is active against MDR-TB and allows shortening of the treatment (GSK-TB Alliance
Fact Sheet http://www.tballiance.org/specials/gsk/gsk-tba_fact_sheet.html; Global TB Alliance
Annual report 2004-2005 http://www.tballiance.org/downloads/2005%20annual%20008_6b.pdf).

Dipiperidine SQ-609 (Sequella Inc.)
Dipiperidine SQ-609 is a novel compound structurally unrelated to existing anti-TB drugs. It kills
M. tuberculosis by interfering with cell wall biosynthesis (precise mechanism unknown). Anti-

16

microbialactivity

has

been

demonstrated

in

vivo

in

mouse

models(http://www.sequella.com/pipeline/SQ%20609.asp; (Nikonenko et al., 2004); (Kelly et al., 1996)

ATP Synthase Inhibitor FAS20013 (FASgene)
FAS20013 is a novel compound identified by Fasgen. It belongs to the class of sulphonylcarboxamides. Fasgen claims that “FAS20013 will kill more organisms in a 4-hour
exposure than isoniazid or rifampicin can during a 12- to 14-day exposure. The compound is very
effective in killing MDR-TB organisms that are resistant to multiple drugs currently in use. A seriesof recent laboratory experiments indicates the superior effect of FAS20013 compared to current
drugs in terms of its ability to "sterilize" TB lesions and kill latent TB. Therapeutic evaluation of
FAS20013 has repeatedly shown its effectiveness in mice, and appears to have no serious side
effects. The compound is up to 100% bioavailable when administered orally. To date no doselimiting toxicity has been encountered, even when doses 10 times the effective dose wereadministered.” The compound is thought to act through inhibition of ATP synthase. However,available scientific publications assessing the efficacy of this compound are of poor quality. (Joneset al., 2000; Parrish et al., 2004)

Translocase I Inhibitor (Sequella Inc.)
Sequella is developing a series of translocase inhibitors for the potential treatment oftuberculosis. The compounds specifically inhibit mycobacterial translocase I, an enzyme requiredfor bacterial cell wall synthesis. Preclinical evaluation of the compounds is planned.
(http://www.sequella.com/pipeline/translocaseinhibitor.asp.).

InhA Inhibitors (GlaxoSmithKline-TB Alliance)
InhA, the enoyl reductase enzyme from Mycobacterium tuberculosis, catalyses the last step inthe fatty acid biosynthesis pathway (FAS II). Frontline anti-tuberculosis drugs such as isoniazid(INH) target this enzyme. Drug resistance to INH results primarily from mutations in KatG, theenzyme that activates INH. Consequently, InhA inhibitors that do not require activation by KatG are
attractive candidates for drug discovery. The main purpose for this screen is therefore to bypass
the activation step and directly inhibit InhA. A possible limitation for this kind of compounds is thatcross-resistance with isoniazid may easily occur. Indeed, mutations in InhA encoding gene havebeen already identified in INH-resistance strains (Banerjee et al., 1994) even if they occur lessfrequently than KatG mutations.

Isocitrate Lyase Inhibitors (GlaxoSmithKline-TB Alliance)
The isocitrate lyase (ICL) enzyme has been shown to be essential for long-term persistence ofM. tuberculosis in mice, but not required for bacilli viability in normal culture or hypoxic conditions
17

(McKinney et al., 2000). McKinney and collaborators have recently shown that inhibition of ICL1
and ICL2, the two isoforms of isocytrate lyase present in M. tuberculosis, blocks growth andsurvival of M. tuberculosis bacteria in macrophages and in mice at early and late stage of infection(Munoz-Elias and McKinney, 2005). The absence of ICL orthologs in mammals should facilitate thedevelopment of glyoxylate cycle inhibitors as new drugs for the treatment for tuberculosis. Such a
new drug is expected to be able to kill persistent bacteria and therefore have sterilizing activity andshorten treatment time. Guided by the three-dimensional structure of isocitrate lyase (Sharma et al.,2000), GSK launched in 2000 a screening to identify ICL inhibitors as potential therapeutic drugs.
Up to now 900,000 compounds have been screened but no successful inhibitors have been
identified. GSK is currently planning to screen additional 400,000 compounds (Global TB Alliance
Annual report 2004-05; http://www.tballiance.org/downloads/2005%20annual%20008_6b.pdf). The
structure of ICL active site is making the screening for inhibitors particularly lengthy and laborious.
The active site of this enzyme, indeed, appears not to be easily and effectively reached bycompounds (J McKinney personal communication).

4.2 Compounds originating from existing families of drugs

Using existing Fluoroquinolones for TB ?
Fluoroquinolones were introduced into clinical practice in the 1980s. Characterized by broadspectrum antimicrobial activity, they are recommended and widely used for the treatment ofbacterial infection of the respiratory, gastrointestinal and urinary tracts (Bartlett et al., 2000; Neu,
1987). Fluoroquinolones have been also found to have activity against M. tuberculosis (Grosset,
1992; Tsukamura et al., 1985) and are currently part of the recommended regimen as second-line
drugs (Crofton, J., P. Chaulet, D. Maher, J. Grosset, W. Harris, N. Horne, M. Iseman, and B. Watt.
1997 Guidelines for the management of drug-resistant tuberculosis WHO, Switzerland). Since
fluoroquinolones share the same molecular targets (for details refer to footnote) 2 , it is highly

2

Fluoroquinolones act by inhibiting DNA topoisomerase IV and DNA gyrase, enzymes that control DNA
topology and are vital for cellular processes that involve duplex DNA, namely replication, recombination andtranscription (Lewin CS et al., 1991; Willmott et al., 1994; for a review see Hooper, 2001). By inhibiting these
enzymes, fluoroquinolones block DNA replication and induce DNA damage, triggering a set of still poorly
defined events, which result in eventual cell death. Fluoroquinolone-dependent inhibition of RNA synthesis,
and as a consequence protein synthesis, is also thought to contribute to the bactericidal activity of this classof drugs (Lewin et al., 1991; Willmott et al., 1994). Unlike most other bacterial species, M. tuberculosis lacks
genes encoding for topoisomerase IV as revealed by the full genome sequence (Cole et al., 1998).
Therefore, the main molecular target for fluoroquinolones in M. tuberculosis is the DNA gyrase (Onodera etal., 2001;Aubry et al., 2004). Consistently, resistance to fluoroquinolones in clinical isolates of M.
tuberculosis occurs primarily due to mutations in the quinolone resistance determining region (QRDR) of thegyrA gene, which encodes for the A subunit of DNA gyrase (Sullivan et al., 1995; Kocagoz et al., 1996).
Other mechanisms such as mutations in the B subunit of DNA gyrase, decreased cell permeability to thedrug, and an active drug efflux pump mechanism could also be involved in triggering resistance. In particular,
the expression or overexpression of energy-dependent efflux pumps that can actively remove antibacterial
agents from the cell have been shown to play a role in determination of fluoroquinolone resistance (Li et al.,
2004; Zhanel et al., 2004;Brenwald et al., 1998;Colangeli et al., 2005)

18

probable that they will trigger the same mechanisms of resistance. Indeed, cross-resistance has
been reported within the fluoroquinolone class such that reduced susceptibility to one
fluoroquinolones likely confers reduced susceptibility to all fluoroquinolones (Alangaden et al.,
1995; Ruiz-Serrano et al., 2000); for review see (Ginsburg et al., 2003a). The major concern is thatwidespread use of fluoroquinolones for treatment of other bacterial infections may select forresistant strains of Mycobacterium tuberculosis. Fluoroquinolones susceptibility is not routinely
assessed in clinical isolates of tubercle bacilli, so there is not much information available about theprevalence of fluoroquinolone resistance in M. tuberculosis. In a study conducted in USA and
Canada, among referral samples isolates between 1996 and 2000, resistance to ciprofloxacin wasassessed and was found to occur in 1.8% (33/1852) of isolates. Of those, 75.8% (25/33) were also
multi-drug resistant. The authors concluded that despite the widespread use of fluoroquinolones fortreatment of common bacterial infection in USA and Canada, resistance to fluoroquinolones
remains rare and occurs mainly in multi-drug resistant strains (Bozeman et al., 2005). In contrast,
in a different study conducted by Ginsburg and collaborators at the John Hopkins Hospital(Baltimore) between 1998 and 2002, the incidence of M. tuberculosis fluoroquinolone resistance ina small sample of patients (55) with newly diagnosed tuberculosis was found to be high among
patients with prior fluoroquinolone exposure (2/19). Cross-resistance was observed among thedifferent

fluoroquinolones

tested(ofloxacin,

levofloxacin,

gatifloxacin, moxifloxacin,andciprofloxacin) (Ginsburg et al., 2003b). In the Philippines, where fluoroquinolone use is poorly
controlled, among 117 TB patients, fluoroquinolone resistance occurred in 53.4% of those with M.
tuberculosis resistant to 4 or 5 drugs, in 23.3% with resistance to 3 drugs and in 18.2% withresistance to 2 drugs (Tupasi TE MDR in the Philippines 4th World Congress on TB, Washington
DC2002). Because of the high prevalence of Tuberculosis in the Philippines, fluoroquinolones are
not included in the clinical practice guidelines for the treatment of community-acquired pneumonia(The Philippine clinical practice guidelines on the diagnosis, empiric management and preventionof community-acquired pneumonia in immunocompetent adults. Report of the Task Force on
Community Acquired Pneumonia. Manila: The Philippine Practice Guideline Group–Infectious
Diseases, 1998. 1: 1–29). In conclusion, there are reasons for concerns about the rapid
development of resistance particularly when fluoroquinolones are administered as the only active
agent in a failing multi-drug regimen ((1992)Hong Kong Chest Service /British Medical ResearchCouncil. A controlled study of rifabutin and an uncontrolled study of ofloxacin in the retreatment ofpatients with pulmonary tuberculosis resistant to isoniazid, streptomycin and rifampicin (Alangaden
and Lerner, 1997; Yew et al., 1990). Moreover, the risk of selecting fluoroquinolone-resistant M.
tuberculosis strains by empirically treating with fluoroquinolones other presumed infections before
a diagnosis of tuberculosis is established is of great concern. For this reason some investigators in
the TB field argue that the use of fluoroquinolones might be better reserved for specific serious

19infection such as tuberculosis rather than becoming the workhorse of antimicrobial treatment; .
however, given the current widespread use of quinolones this might not be realistic.
Lately, the interest on fluoroquinolones as antituberculosis agent has focused on the new
fluoroquinolones moxifloxacin (MXF) and gatifloxacin (GTF). Despite a lack of a comprehensive
work comparing the activities of old and new classes of Fluoroquinolones in M. tuberculosis, what
can be inferred from published results is that moxifloxacin and gatifloxacin are characterized by ahigher activity against M. tuberculosis in vitro when compared to the old fluoroquinolones ofloxacin
and ciprofloxacin (Hu et al., 2003; Paramasivan et al., 2005; Rodriguez et al., 2001; Sulochana etal., 2005). These new compounds are currently taken in consideration as anti-tuberculosis first-line
drugs. A more detailed analysis of the properties of the new fluoroquinolones moxifloxacin andgatifloxacin will follow in the next paragraphs.

Gatifloxacin. Marketed in the U.S. by Bristol-Myers Squibb as Tequin, Gatifloxacin (GAT) has
been found to have in vitro and in vivo bactericidal activity against M. tuberculosis ((Hu et al.,2003);(Alvirez-Freites et al., 2002)). In an in vitro study using stationary-phase mycobacterial
culture, gatifloxacin (4 μg/ml) showed the highest bactericidal activity during the first 2 days but notthereafter (Paramasivan et al., 2005). Similar results were obtained when gatifloxacin was used in
combination with isoniazid or rifampicin: gatifloxacin was able to slightly increase the bactericidal
activity of INH or RIF only during the first 2 days (Paramasivan et al., 2005). This is in contrast withother studies showing that gatifloxacin and moxifloxacin had similar bactericidal activity on a
stationary-phase culture of M. tuberculosis and comparable to the bactericidal activity of rifampicin(Hu et al., 2003; Lenaerts et al., 2005). One paper reported that when tested in mice in
combination with Ethionamide and Pyrazinamide (high doses: 450 mg/kg, 5 days per week)
gatifloxacin was able to clear the lungs of infected animals after 2 months of treatment (Cynamon
and Sklaney, 2003). Thus, currently available data on gatifloxacin do not support the hypothesisthat introduction of gatifloxacin in first-line regimen will impressively contribute to shorten TB
treatment. Further investigation should be addressed to properly assess the activity of gatifloxacin
in vitro and in mouse models.
Nevertheless, gatifloxacin is currently in Phase III Clinical Trials, conducted under thesupervision of the European Commission Oflotub Consortium, WHO-TDR, NIAID TBRU,
Tuberculosis Research Centre. The aim of the trial is to evaluate the efficacy and safety of a fourmonths gatifloxacin-containing regimen for the treatment of pulmonary tuberculosis.

Moxifloxacin. Produced by Bayer Pharmaceuticals and marketed as Avelox in the USA,
moxifloxacin is the most promising of the new fluoroquinolones being tested against M.
tuberculosis.

20In vitro, moxifloxacin appeared to kill a subpopulation of tubercle bacilli not killed by rifampicin, i.e.
rifampicin-tolerant persisters, while the older fluoroquinolones ciprofloxacin and ofloxacin did nothave any significant bactericidal effect on the same subpopulation (Hu et al., 2003). One possibility
is that moxifloxacin interferes with protein synthesis in slowly metabolising bacteria through amechanism that differs from that used by rifampicin. However, the molecular mechanisms beyond
such a bactericidal activity still await further characterization. (Refer to footnote for otheradvantages at molecular level of moxifloxacin in comparison with older fluoroquinolones, i.e.
ciprofloxacin and ofloxacin)3 .
In mouse models the activity of moxifloxacin against tubercle bacilli was comparable to that ofisoniazid (Miyazaki et al., 1999). Moreover, when used in combination with moxifloxacin andpyrazinamide, moxifloxacin has been reported to kill the bacilli more effectively than theisoniazid+rifampicin+ pyrazinamide combination. Indeed, cultures from lungs of mice treated withrifampicin-moxifloxacin-pyrazinamide for 2 months followed by rifampicin-moxifloxacin resulted
negativeupon

4monthsoftreatment,

while

mice

that

receivedrifampicin-isoniazid-

pyrazinamide/rifampicin-isoniazid showed complete culture conversion after 6 months of treatment(Nuermberger et al., 2004a). Furthermore, no relapse was observed in mice treated for at least 4months with the combination rifampicin-moxifloxacin-pyrazinamide, while mice treated withrifampicin-isoniazidpyrazinamide required 6 months of treatment before no relapse could be
detected (Nuermberger et al., 2004a); (Nuermberger et al., 2004b). The authors explain the better
activity of the rifampicin-moxifloxacin-pyrazinamide combination over the rifampicin-INHpyrazinamide combination as the consequence of a possible synergism in the anti-tuberculosis
activity of the three drugs rifampicin, moxifloxacin and pyrazinamide. Alternatively, substitution of
moxifloxacin with isoniazid in the standard regimen could relieve a possible antagonism among thecurrently used drugs (Grosset et al., 1992).
To summarise, results obtained so far in in vitro and in vivo studies suggest that moxifloxacin
might be a promising candidate drug to shorten TB treatment. At the molecular levels, the reason
for its improved efficiency is mainly a consequence of its poor susceptibility to active efflux that
ensures the maintenance of high intracellular concentration. Shortening of therapy in mousemodels seems to be mainly due to a released of antagonism among the drugs in the regimen.
There is a possibility that moxifloxacin might be active against slowly metabolising bacteria by

3-Moxifloxacin appears to be a poor substrate for efflux pumps in other pathogens such as S. pneumonia.
This might be due to the bulky C-7 substituent that characterizes this compound. In contrast older and morehydrophilic fluoroquinolones such as ciprofloxacin are more susceptible to be actively exported outside thebacterial cell (Daporta et al., 2004);(Coyle et al., 2001),(Pestova et al., 2000)-In vitro, moxifloxacin inhibitory activity of DNA gyrase is higher than ofloxacin but comparable tociprofloxacin (Aubry et al., 2004).
-In S.pneumoniae, moxifloxacin maintained clinically useful level of activity against bacterial strains that bore
mutations in the QRDR region of gyrA genes, suggesting that MXF could target other key domains in the
DNA-gyrase enzyme (Pestova et al., 2000)
21

inhibiting DNA transcription and, consequently, mRNA and protein synthesis, therefore having amild sterilizing activity. However, this still needs to be rigorously proven.
As far as emergence of resistance is concerned, in vitro studies in S.pneumoniae revealed thatmoxifloxacin, used at concentration above the minimal inhibitory concentration (MIC), is less prone
to select first-step mutants when compared to the fluoroquinolone sparfloxacin. However,moxifloxacin monotherapy in mice models showed that resistance to moxifloxacin might rapidly
emerge (Ginsburg et al., 2005).
Moxifloxacin is currently in Phase II Clinical Trials. A trial substituting ethambutol withmoxifloxacin during intensive phase (TBTC 27) was initiated before above cited animal studies had
been conducted and showed no advantage over ethambutol. Preliminary results of this study havebeen published and showed that moxifloxacin containing regimen did not present increased
sterilizing activity (measured as the ability to induce sputum colture conversion upon 2 month oftreatment) over the standard regimen. However moxifloxacin-containing regimen did show
increased activity at earlier time points (Burman et al., 2006). A new trial (TBTC 28), substituting
moxifloxacin with isoniazid in intensive phase to compare culture conversion rate will be carried out
by CDC in cooperation between TB Alliance and Bayer in several sites in the US as well as sites in
Spain, South Africa and Uganda. At time of writing this report the trial is recruiting.
(http://clinicaltrials.gov/ct/gui/show/NCT00144417?order=4;

http://www.tballiance.org/bayer/docs/english-TB_Alliance_Bayer _Release.pdf)..

New Quinolones
In 2003 the TB Alliance launched a project in collaboration with the Korean Research Institute ofChemical Technology (KRICT) and the Yonsei University aimed to synthesize and evaluate noveland more effective quinolone compounds that could shorten first-line treatment. To date, 450
compounds have been synthesized and tested for their anti-TB activity. During this work, the subclass termed 2-pyridones has been identified as the one showing most potent activity against M.
tuberculosis in both its growing and persistant state. This sub-class of compounds was alreadyidentified by Abbott in 1998 and found to have activity against drug-susceptible and drug resistant
M. tuberculosis (Oleksijew et al., 1998). In June 2005, the TB Alliance contacted Abbott and was
granted rights to develop for TB indication this class of DNA gyrase inhibitors which is otherwise
protected by certain Abbott patents. As fluoroquinolones, 2-pyridones are inhibitors of DNA gyrase(Flamm et al., 1995). Current work is focused on the modification of a position which influences
activity, pharmacokinetics and safety profile. The lead compounds identified so far showed better
activity than gatifloxacin and moxifloxacin. At present, the project is in the lead optimisation stage
and aims to obtain a final candidate by the end of 2006 (TB Alliance Annual report 2004/2005,
http://www.tballiance.org/downloads/2005%20annual%20008_6b.pdf)

22Non-fluorinated quinolones
Recently, a series of 8-methoxy non-fluorinated quinolones (NFQs) have been developed by
Procter & Gamble. NFQs lack a 6-fluorine in their quinolone nucleus differentiating them fromfluorinated quinolones such as gatifloxacin and moxifloxacin. NFQs target a broad spectrum ofbacteria and they seem to act preferentially through inhibition of DNA gyrase (Barry et al., 2001;
Jones et al., 2002). NFQs are currently being tested against M. tuberculosis.

Diamine SQ-109
Diamine SQ-109 has been identified in a screening performed by Sequella Inc. using a
combinatorial library based on the pharmacophore of ethambutol. The aim was to develop asecond-generation agent from the first line drug ethambutol. When tested in mice using a low-dose
infection model of TB, SQ-109 at 1 mg/kg was as effective as ethambutol at 100mg/kg. However
SQ-109 did not show improved effectiveness at higher doses (10mg/kg; 25mg/kg) and was clearlyless effective than isoniazid (Protopopova et al., 2005). Protopopova and collaborators claim that
SQ-109 is effective against drug-resistant strains of M. tuberculosis, including those that are
ethambutol-resistant, and that it targets different intracellular target(s). For this reason it can beconsidered as a new TB drug and not simply as an ethambutol analogue.

Macrolides
The aim of this project, launched by the TB Alliance in collaboration with the Institute for TB
research of the University of Illinois in Chicago, is to optimise the anti-TB activity of the macrolide
antibiotics through the synthesis of additional chemically modified derivatives of erythromycin.
More than 200 derivatives have been synthesized and three series were identified as having antituberculosis activity superior to that of the benchmark clarythromicin. Members of these series
have exhibited potent anaerobic activity and appears to be safe in use with ARVs (TB Alliance
Annual report 2004/2005, http://www.tballiance.org/downloads/2005%20annual%20008_6b.pdf).

Thiolactomycin analogs
Thiolactomicin (TLM) was the first example of naturally occurring thiolactone to exhibit antibiotic
activity. The compound has moderate in vitro activity against a broad spectrum of pathogens,including Gram-positive and Gram-negative bacteria and M. tuberculosis. Analogs of thiolactomicin
have been synthesized and found to have enhanced activity against whole cells of pathogenic
strain of M. tuberculosis (Douglas et al., 2002). TLM analogs seem to act through the inhibition of
the mycolate synthase, an enzyme involved in the biosynthesis of the cell wall.

23

Nitrofuranylamides
M. tuberculosis is quite susceptible to Nitro-containing compounds (Sun and Zhang, 1999)(Murugasu-Oei and Dick, 2000). Nitrofuranylamide was identified in a screening for UDP-Gal
mutase inhibition. An expanded set of nitrofuranylamides was synthesized and tested for antimicrobial activity. This led to the identification of a number of nitrofuranylamides with activity
against M. tuberculosis. However, the further investigation has revealed that the primary target for
nitrofuranylamides antimicrobial activity is not the UDP-gal mutase. Four compounds of thenitrofuranylamides class showed significant activity in mouse models for TB infection(Tangallapally et al., 2004)

Nitroimidazole Analogs
While pursuing PA-824’s remaining development activities, the TB Alliance in collaboration with
the Novartis Institute for Tropical Diseases initiated in 2004 a back-up program to maximize thepotential of this class of compounds by identifying superior compounds and improving on PA-824’s
properties as a drug.

Moreover, a number of drug screenings and target identification screenings are underway atseveral research institutes and pharmaceutical companies, notably GlaxoSmithKline (Tres Cantos,
Spain) and AstraZeneca (Bangalore, India).
Several public research institutes (University of Auckland, National Institute for Allergy and
Infectious diseases, California State University, Institute for tuberculosis Research-University of
Illinois) are screening libraries of natural products (plant and bacterial products) with the hope toidentify compounds that have anti-tubercular activity.
Dr Carl Nathan at the Cornell University Weill Medical College received a $3.5 million grant fromthe National Institute of Allergy and Infectious Diseases (September 15, 2004 through August 31,
2007) to carry out a project aimed to the characterization of the M. tuberculosis enzyme
Dihydrolipoamide

Acyltransferase

asapotential

targetforchemotherapy

ofTB(http://www.med.cornell.edu/news/dean/2005/01_05_05/article7.html;
http://www3.niaid.nih.gov/Biodefense/Research/2004awards/03016_awards.htm). M. tuberculosis
Dihydrolipoamide Acyltranferase (dlaT) is a component of two important multi-subunit complexes:
pyruvate dehydrogenase, the enzyme that catalyses the synthesis of Acetyl Coenzyme A, andperoxynitrite reductase, a defence against oxidative/nitrosative stress (Tian et al., 2005); (Shi andEhrt, 2006). DlaT has been shown to be required for full virulence in vivo in mice, while in in vitro
experiments mouse macrophages can readily kill intracellular M.tuberculosis mutants lacking dlaT
(Shi and Ehrt, 2006).
GSK and the TB Alliance are developing several projects under the general category of“Focused screening”. The aim is to identify compounds that are active against specific distinct

24molecular targets, including inhibitors of DNA gyrase (the target of fluoroquinolones), peptide
deformylase (PDF) inhibitors and analogs of quinolone electron transport inhibitors.
Bacterial peptide deformylase belongs to a subfamily of metalloproteases catalysing theremoval of the N-terminal formyl group from newly synthesized proteins. PDF is essential forbacterial growth but is not required by mammalian cells, so represents a promising target for thedevelopment of a new generation of broad-spectrum antibacterial agents. Two PDF inhibitors, VIC104959 (LBM415) and BB-83698, have progressed to Phase I clinical trials (Jain et al., 2005). The
PDF inhibitor BB-3497 was recently found to have potent in vitro activity against M. tuberculosis(Cynamon et al., 2004). This finding suggests that PDF inhibitors can find application in TB
treatment. Beside the project jointly launched by GSK and the TB Alliance, researcher at theNovartis Institute for tropical Disease (NITD) are also working on identification of PDF inhibitors for
TB

treatment(NITD

Symposiumon

Tuberculosis,

October

17-20

2005,

http://www.nitd.novartis.com/includes/teasers/teaser_attaches/tb_program_final.pdf).
Inhibition of electron transport can lead to ATP depletion and decline in intracellular redox
potential. Recently, anti-tubercular drugs targeting ATP synthesis (i.e. diarylquinoline) have beenshown to be particularly effective, even against non-replicating bacteria. Therefore, identification ofcompounds able to inhibit the electron transport process could lead to the development of more
effective drugs active against both replicating and non-replicating bacilli.

Others drugs candidate that could find an application in TB treatment are:
New rifamycin derivatives
Rifalazil, a new semisynthetic rifamycin, is characterized by a long half-life and is more active thanrifampicin and rifabutin against M. tuberculosis both in vitro and in vivo (Hirata et al., 1995; Shoen
et al., 2000). However, high level rifampicin -resistant strains present cross-resistance to allryfamycins (Moghazeh et al., 1996).

Oxazolidinones (Linezolid)
Oxazolidinones are a new class of broad-spectrum antibiotics developed by Pharmacia. They
inhibit protein synthesis by binding to the 50S subunit of ribosomes. Oxazolidinones had significant
activities against M. tuberculosis in vitro and in mice (Cynamon et al., 1999; Zurenko et al., 1996).
However, oxazolidinones are seen as less promising due to their toxicity and high price.

4.3 Summary of the drug pipeline.
Table 3 provides an overview of available information on the main properties of the drugs in thepipeline. (The overall strengths and weaknesses of the current pipeline are further discussed in
section 6).
25

Discovery

Preclinical

ClinicalTable 3. Main properties of candidate anti-TB drugs

DRUG

Effect on
bacteria cells

Mechanism of action

Targets

Activity against MDRTB

Dyarylquinoline
TCM207Bactericidal
Potentiallysterilizing

ATP depletion andimbalance in pH
omeostasis

AtpE, component of
ATP synthase

Active against MDR-TB
NO cross-resistance withcurrent TB drugs

Gatifloxacin

Bactericidal

Inhibition DNA replicationand transcription

DNA gyrase

Active against isoniazid
and rifampicin resistantstrains (weak data)

Moxifloxacin

Bactericidal

Inhibition DNA replicationand transcription

DNA gyrase

Active against MDR-TB in
combination withethionammide (ETH)

Nitroimidazole
PA-824

Sterilizing in vitro
Bactericidal in vivo

No data available

Active against MDR-TB
NO cross-resistance

Pyrrole LL-358

Bactericidal/
Sterilizing(?)

Inhibition of protein
synthesis
Inhibition of cell wall lipids
synthesis
No data available

No data available

Active against MDR-TB

NitroimidazoBactericidal
oxazole OPC-67683

Inhibition of cell wallbiosynthesis

No data available

Active against MDR-TB

Diamine SQ-109

Bactericidal(?)

Inhibition cell wallbiosynthesis

No data available

Effective against
ethambutol-resistantstrains(?)

Dypiperidines (SQ609)

Bactericidal(?)

Inhibition of cell wallbiosynthesis

No data available

No data available

ATP Synthase
Inhibitor FAS20013

No data available

ATP depletion

ATP synthase (?)

In vitro activity against
MDR-TB strains

Translocase I
Inhibitor

Bactericidal (?)

Inhibitor of cell wallbiosynthesis

Translocase I

No data available

Non-FluorinatedQuinolones

Bactericidal

Inhibition DNA replication

DNA gyrase

No data available

Nitrofuranylamides

No data available

No data available

No data available

No data available

Picolinamide
Imidazoles

No data available

No data available

No data available

No data available

Pleuromutilins

Bactericidal/
sterilizing?

Inhibition of protein
synthesis

Large subunit ofribosome

Active against MDR-TB

26

Discovery

Thiolactomycin
Analogs

Bactericidal?

Inhibition of cell wall
bioshyntesis

Mycolate shynthase

No data available

Dihydrolipoamide
Acyltransferase
Inhibitors

No data available

Dihydrolipoamide
Acyltransferase

No data available

InhA inhibitors

Bactericidal (?)

Inhibition of basic
metabolism andoxidative/nitrosative stressresponse
Inhibition of cell wallbiosynthesis

Enoyl ACP
reductase

Prone to cross-resistancewith INH

Isocitrate lyase
Inhibitors (ICL)

Expected to besterilizing

Inhibition of glyoxylate
cycle

Isocitrate lyase

No data available

Methyltransferas
inhibitors

No data available

No data available

No data available

No data available

Quinolones

No data availableexpecetedbactericidal

Inhibition DNA replicationand transcription

DNA gyrase

No data available-prone
to cross resistance withfluoroquinolones

5. Expected timelines towards approval for new candidate drugs
The expected time schedule towards approval for drugs that are currently in clinicaldevelopment is summarised in Figure 2.

Figure 2. Expected timelines towards approval of candidate drugs currently in clinical stage ofdevelopment (Sources: Global TB Alliance Annual report 2004-2005;StopTBPartnership Working Groupon New Drugs for TB. Strategic Plan 2006-2015)27

Clinical trials to register a TB drug represent a lengthy and expensive process that can take aminimum of six years, generally longer than for other infectious diseases. The greatest challenge inthe design of TB clinical trials is in Phase III Trials. These trials are usually large scale, randomised
clinical trials designed to show improvement or equivalent efficacy compared to the standard
regimen among diseased patients. Efficacy evaluation requires measurements of relapse rateduring a 1-2 years follow-up after completion of the already lengthy 6 months treatment regimen.
Relapse rate after chemotherapy is commonly accepted as the endpoint to determine the efficacy
of a new therapy and to assess whether a new drug can improve sterilising activity. Since relapse
rates under random clinical trial conditions are often 3% or less, large numbers of patients areneeded to demonstrate an improvement in relapse rate. This results in high drug development
costs and long delays in introducing new medicines.
Validated surrogate markers of relapse would provide evidence on the efficacy and thesterilising activity of a drug/regimen without requiring large numbers of patients in a conventional
clinical trials and with great savings in development time and cost. The best validated surrogate
marker for relapse is the proportion of sputum cultures that remain positive after about 2 months ofchemotherapy. This method has been shown to associate with the fall in relapse rates in 8 clinical
trials (Mitchison, 1993; Mitchison, 1996). Less well validated procedures that require further studies
and validation are the rate of sputum conversion (Durban Immunotherapy Trial Group, 1999), themeasurement of the 85B (alpha) antigen of M. tuberculosis in sputum (Desjardin et al., 1999) andthe extended studies (beyond 2 days) of early bactericidal activity (EBA) (Sirgel et al., 2000).
However, regulatory agencies still require that drug’s efficacy is demonstrated during phase III
trials through a combination of traditional and surrogate markers for activity.
Since the identification of biomarkers could significantly streamline and accelerate clinicaldevelopment, the TB Alliance has recently established a collaboration with BG Medicine Inc. toidentify biomarkers for drug efficacy in TB treatment. A biomarker is a quantifiable biochemical
characteristic (such as a metabolite, hormone or enzyme) that is measured and evaluated as apharmacologic response to chemotherapy. The TB Alliance/BG Medicine project will aim to identify
biomarkers for two purposes: (i) to provide an early indication of drug’s ability to shorten treatment
during Phase II testing; (ii) to act as a surrogate marker of treatment efficacy and sterilizing activity
that could shorten Phase III trials and eliminate the need for the 2 years follow-up to determine
relapserates(http://www.tballiance.org/pdf/PRESS%20Release%20TB%20Alliance-

BG%20Medicine%20FINAL.pdf).
The current need for large number of patients recruited for Phase III clinical trials implies theneed to conduct trials in countries with high TB incidence rates in order to ensure that a sufficientstudy population can be obtained. One of the major challenges that the TB Alliance and Pharmacompanies are facing while embarking in clinical development concern the lack of clinical trial
capacity in endemic countries, Phase III infrastructure is in particular poorly developed and might28

require additional coordination, regulatory support and specific funding to overcome gaps. In order
to be considered as potential sites to run clinical trials for drug approval Good Clinical Practicestandards (GCP) need to be implemented. The TB alliance faces also difficulties with regulatory
issues because regulatory authorities (FDA, EMEA) have no experience any longer in approving
new TB drugs (TB Alliance personal communications).

6. A crucial gap: lack of early stage drug discovery
As described in Appendix A, several genes that could be important for survival of M.
tuberculosis in vivo have been identified and characterized during the last years. But validation of
those potential drug targets through genetic or chemical inactivation is still largely missing. As
pointed out by Dr Valerie Mizrahi (University of the Witwatersrand, Johannesburg) “there is anurgent need to put in place the advanced knowledge that we gained about M. tuberculosis
metabolism and physiology and think how to translate it into validated target that can be used forscreening of new drugs”.
Why is this not happening? Scientists working in the TB field have identified primarily two kindof obstacles that are hindering the drug discovery process. On one side there are “scientificobstacles” primarily represented by the current inadequate characterization of TB lesions in humanhost. On the other side there are “financial obstacles” mainly represented by the lack of interest by
current industry initiatives, lack of capacity by the TB Alliance to enter into this field, and lack ofsustained funding for academic laboratories to run research projects that fall in the borderline
between basic and applied science
A systematic characterization of the heterogeneity of TB lesions in patients aimed to get aclearer picture of the different microenvironments that bacteria have to adapt to in order to survive
and persist in human hosts, is still largely missing. Similarly, the understanding of the criticalmechanisms that underlie survival of M. tuberculosis during the extended periods of chemotherapy
is still rather limited. These gaps in our knowledge of M. tuberculosis biology are making theidentification and validation of potential targets that are relevant in vivo in the human host still arather difficult task. The “biological uncertainties” about M. tuberculosis also represent one of thereasons why pharmaceutical companies consider anti-TB drug discovery and development as
particularly risky ground and are therefore generally reluctant to embark on this kind of projects.
These critical open questions will be partially addressed by a research project coordinated by Dr
Douglas Young (Imperial College London) and funded by the Gates Foundation as part of theGrand

Challenges

in

Globalhealthinitiative(GrandChallenge

#11,

June

2005;

http://www.gcgh.org/subcontent.aspx?SecID=403). In an effort to develop drugs for latent TB, thisinternational collaboration is aiming to identify the molecular pathways essential for the bacteria to29

survive inside human tissues. The purpose is to select targets that are essential for viability of"persistent" bacteria that are relatively tolerant to conventional drugs.
Another “scientific obstacle” that is currently under debate is the lack of adequate animalmodels truly representative of human latent tuberculosis. Although there is a consensus that mice,
rabbits, guinea pigs and non-human primates infected with M. tuberculosis can model overlapping
characteristics of human TB, some scientists strongly advocate for the use of non-human primatesas the animal model of choice (Boshoff and Barry, 2005; Flynn and Chan, 2001). Currently, themost popular model is the murine model due to low cost, availability of genetically defined strains
and comprehensive characterization of mouse immunology. While there are similarities in the
immune control of TB in mice and humans, the progression of the disease is markedly different. In
particular the features of chronic infections in mice (high bacterial titre in the lungs and spleen,absence of necrotic lesions, precursors of cavities) do not reflect the situation of advanced TB in
humans (Gupta and Katoch, 2005; McMurray et al., 1996). However, the choice of the “right”
animal model requires a previous systematic characterization of TB lesions in the human hosts. As
far as this characterization will be largely missing it might be premature and is not really evidencebased to advocate for one or another animal model.
Lack of sustained funding for early stage drug discovery projects is the other barrier in theprocess of anti –TB drug discovery. Examples are target validation projects or projects in the fieldof chemical genetics. Up to now major funding bodies for TB research are represented byorganisms such as NIH/NIAID, Wellcome Trust and EU. These funding organisms and the peer
review system that they use to select grant recipients traditionally give priority to “blue-skies” basic
research projects and “hypothesis-driven” science. Several experts in the field point out thatexceptions made for specific grants based on request for application (RFA), it is pretty difficult for
academic labs to obtain funds for projects that fall in between basic and applied research.
The fact that this kind of projects falls into cracks is creating a serious gap in the drug discovery
process. The pharmaceutical industry considers it economically risky to develop drugs for diseases
such as TB that are endemic in developing countries and do not offer exciting market perspectives.
While pharmaceutical companies actively scout for advances in basic science and are ready toinvest in early stage drug discovery in areas such as cancer drug development, the new TB
programs by pharmaceutical companies are not doing this and are still to timid and risk avert. As
pharmaceutical companies want their risk to be reduced to minimum levels when they embark on
projects aiming to develop drugs for neglected diseases, they are more likely to become interested
and accept to run a drug development project when rigorously validated targets are available orwhen a lead compound has been already identified. “As a consequence, more effort is required toacademic labs working on TB, that are asked to provide target validation and/or drug leads” said Dr
Guilhot (CNRS, Toulouse) and Dr Sherman (Univeristy of Washington, Seattle). So far, the TB
Alliance, despite all its pioneering work, has not entered the area of translational research either,30

likely because of financial reasons as they are still working to even secure funding for their existing
projects.
What happens then if early stage drug discovery projects cannot be carried out in academic
laboratories? “In TB field, target identification and target validation projects are not carried out bybiotech or pharmaceutical companies due to lack of interest and lack of expertise - there is little
capacity and expertise on TB among pharmaceutical companies since there have not beenongoing TB development projects. If this work cannot be done by academic laboratories, theprocess gets stuck” said Dr John McKinney (Rockefeller University, New York). It is worth notingthat a research project aimed to target validation through genetic knockout is an extremely focused
project. In an academic lab that has well-established expertises and facilities, the work can bedone relatively quickly and with a limited amount of money. If faster progress is to be achieved“there is the need to enable the academic sector to go beyond the proof of principle thattraditionally is the final aim”, said Dr Mizrahi. As pointed out by Dr Nathan, “it is necessary to rethink the traditional roles played by academia and pharmaceutical industry in drug discovery anddevelopment and push academia into fields that are traditionally ground for industry when it comesto drugs for diseases that do not ensure appealing market perspectives”. For this to happen,
focused funding streams need to be established for translational research projects.
NIH/NIAID tried to make things move in this direction by establishing and funding facilities suchas TARGET (Tuberculosis Animal Research and Gene Evaluation Taskforce) and TAACF(Tuberculosis Antimicrobial Acquisition and Coordinating Facility) that offer services respectively
for testing of M. tuberculosis mutants in animal models and for high-throughput screening of large
compound libraries against validated target. However this strategy does not look to be extremely
efficient and successful so far. While this is a step in the right direction, it is far from satisfactory. It
is likely that pull programs, which support projects on the basis of achievement rather than pushprograms supporting classical types of proposals, would be better suited to achieve this goal.
Progress in the field of drug development arising from close collaborations between theacademic sector and pharma companies is promising. Despite the current industry stagnation in
antibiotic R&D, recent research by two industry–academic collaborative groups has resulted in the
discovery of two new classes of antimicrobial peptides for treating Gram-positive bacterial
infections (Brotz-Oesterhelt et al., 2005; Mygind et al., 2005). So far, the only example of such kindof collaboration in the field of TB known to us is represented by the work done on the M.
tuberculosis enzyme isocitrate lyase (ICL). This work has seen a coordinated collaboration among
the laboratories of Dr McKinney (Albert Einstein College of Medicine, Rockfeller University), Dr
Sacchettini (Texas A&M University), Dr Russell (Washington University School of Medicine), andGlaxoSmithKline. In this example the academic sector provided a package that comprised target
validation (Dr McKinney), enzyme crystal structure (Dr Sacchettini) and establishment of thebiochemical assay needed for the high-throughput screening (Dr Russell). GSK is now performing

31

the high-throughput screening for the identification of ICL inhibitors. More recently, this project has
seen the added involvement of the TB Alliance and is now being directed under the collaboration
between GSK and the TB Alliance. The work in academic laboratories was funded by GSK,
through a grant of the “TB Action Initiative” that currently has been completed. While suchacademic-industry cooperation can be successful scientifically, it is important to follow through toensure that the fruits of such research lead to products, even if these should be less profitable, andthat equitable access to the product will be guaranteed.

7. Discussion and conclusions
As new models of drug development are being established, major advances have beenachieved in the basic research field. Modern molecular and genetic tools have become available
for M. tuberculosis (i.e. targeted mutagenesis, array-based analysis of mutant libraries, techniques
for conditional gene silencing) and this has led to impressive improvements in the knowledge andunderstanding of the fundamental biology and physiology of M. tuberculosis (Jansen and Yu, 2006;
Kana and Mizrahi, 2004; Kaufmann et al., 2005; please refer to Appendix A for a detailed review ofmajor advances achieved in the basic research field). This was made possible by funding
programmes supportive for research on M. tuberculosis lunched by major research fundingorganisms (NIH/NIAID, Wellcome Trust, EU) during the 1990s. However, as testified by theacademic community, the situation is changing again and there are serious concerns that such asustained funding availability will not last for long.
Today the TB drug pipeline is richer than it has been in the last forty years. This is also thanks
to the work carried-out by the Global Alliance for TB Drug Development which is associated withapproximately half of all compounds (or projects aimed to identify candidate compounds) currently
being developed. Increased public awareness on the lack of R&D for neglected diseases in recent
years has also led some multinational pharmaceutical companies to invest in TB drug development
on a ‘no-profit-no-loss’ basis (namely, Novartis, AstraZeneca and GlaxoSmithKline). Some
pharmaceutical companies have engaged in tuberculosis R&D on a commercial basis, and withsome success:three of the six anti-TB candidate drugs currently in clinical trials have beendeveloped for profit.

Pressing needs still remain
Despite the positive changes occurred in the last years, there are still problems that need to be
tackled and major roadblocks still exist that are hindering the implementation of rational drug
design and a fast progress in anti-TB drug R&D.
A first important question is if there are enough promising compounds in the TB pipeline for acomprehensive new TB treatment to be developed (Glickman et al., 2006). Although different
32attrition rates might apply, the number of candidate compounds is still small if compared to drug
pipeline for diseases that principally affects wealthy countries. This is reflected by the limited
number of biotech and pharmaceutical companies working on TB (see Figure 3). The ambition of
the TB Alliance and its partners is to register an improved, faster acting regimen by 2010 and aregimen containing completely novel drugs by 2015 (TB Alliance Annual report 2004-2005
http://www.tballiance.org/downloads/2005%20annual%20008_6b.pdf). It is not possible to say
today if the current pipeline will permit the attainment of this goal.

Figure 3. Comparison of drug pipelines for TB and for “more profitable” diseases.
Graphs represent the number of drugs in clinical stage of development (A) and number of pharmaceuticaland biotech companies involved in drugs development projects (B) for Tuberculosis, Cancer andCardiovascular diseases. Source: Pharmaceutical Research and manufacturers of America (PhRMA)
Survey (http://www.phrma.org/). (C) Comparison of worldwide burden of disease in DALY (Disability
Adjusted Life Years) for Tuberculosis, cancer and cardiovascular diseases. Source: WHO, World HealthReport 2004 (http://www.who.int/whr/2004/en/09_annexes_en.pdf). (E) Table summarizing the data plottedin the graphs.*Source: PhRMA; §Source: WHO)

Another important issue concerns characteristics of candidate compounds currently indevelopment. Table 4 summarizes the main properties in terms of mechanism of action of thecandidate drugs that are in the pipeline (for more detailed information on single compounds see
also table 3).
It is evident that many of the candidate drugs are either derivatives of existing compounds ortarget the same cellular processes as drugs currently in use. While analogs and derivatives are far
quicker to develop, agents identified by this approach may have cross-resistance problem, as seen
for the new rifamycins or quinolones (Ginsburg et al., 2003a; Moghazeh et al., 1996).
Fresh approaches and novel sets of microbial targets need to be taken in consideration. One
example of a promising new compound is diarylquinoline TMC-207(currently in phase IIa clinical
trials) which acts through a novel molecular mechanism, most probably by inhibiting the ATPase33

synthase, leading to ATP depletion and pH imbalance. Preliminary results in animal models
indicate that it has the potential to shorten the treatment to 2 months (Andries et al., 2005).
What also comes to light from a critical analysis of the drug pipeline is that rational approaches are
weakly implemented in anti-TB drug discovery and development. Even the most promising novel
drug candidates currently in clinical stage were identified serendipitously in screenings that were
not designed originally for activity against M. tuberculosis. Moreover these compounds wereselected for their ability to kill actively growing bacteria (Andries et al., 2005; Stover et al., 2000).
There is consensus among the TB community that in order to obtain a real breakthrough in TB
therapy and drastically shorten the treatment, there is an urgent need to identify compounds acting
on key targets that are essential for mycobacterial persistence. There is a growing awareness thatdifferent subpopulations of bacteria that vary for their metabolism and growing rate can co-exist in
an infected patient. Novel and more effective drugs should be rationally designed to interfere withmetabolic and physiological strategies used by the bacteria to survive to host immune defences.
An example is the search for inhibitors of the isocitrate lyase, an enzyme that has been proven to
be involved in the “dormancy” response: compounds able to inhibit this enzyme are expected to kill
persistent bacteria. However, most of the compounds in the current pipeline target actively growing
bacteria and so have bactericidal but not sterilizing activity. Therefore, while drugs currently in thepipeline could significantly shorten the treatment to two to three months they are unlikely to lead toa major breakthrough and reduce the treatment to a matter of weeks or days.
Table 4. Adapted from STOP TB Working Group on New drugs. Compounds have been categorized
by the author considering their novelty in structure and mechanism of action

34

A critical obstacle to such rational design is the lack of a comprehensive characterization of
the fundamental biology of mycobacteria as they persist in human tissues. Thus, the identificationand validation of potential targets that are relevant for the survival of the bacteria in vivo still
represent a difficult task. This high degree of uncertainty about biochemical processes andmolecular targets that can be potential target for effective new drugs renders the whole drug R&D
process risky and, therefore, even less attractive for pharma investments. There is urgent need fora better characterization of heterogeneity of TB lesions to obtain a clearer picture of the different
microenvironments in which M. tuberculosis persists. Moreover, there is still a pressing need todecrease the degree of uncertainty about the critical metabolic processes that drugs should target
to achieve sterile mycobacterial elimination. As part of the Grand Challenges in Global Healthinitiative (http://www.gcgh.org/subcontent.aspx?SecID=403) the Bill and Melinda Gates Foundation(Gates Foundation) is funding research into the molecular pathways of persistence, aimed at
identifying novel targets and subsequently run target-based drug discovery programs.
A second critical obstacle to the implementation of rational drug design is the lack of wellvalidated drug targets. The fundamental genetics of M. tuberculosis growth and persistence inanimal models are slowly being unravelled. Several enzymes involved in alternative metabolic
pathways, energy generation, micronutrient acquisition, and survival in activated macrophages aswell as in patient lesions have recently been identified as new sets of potential anti-microbial
targets (Shi et al., 2005; Darwin et al., 2003, Sassetti et al., 2003; Schnappinger et al., 2003;
Rachman et al., 2006), but validation of these potential targets through genetic or chemical
inactivation is largely missing. This creates a critical gap in early stage drug discovery research(figure 4). There is an urgent need to translate this advanced knowledge about M. tuberculosis
metabolism and physiology into validated targets that can be used for screening of new leadcompounds. A key difficulty lies in securing sustained funding for research projects that fall into thearea of target validation and chemical genetics.
The Gates Foundation recently announced a new initiative that specifically aims at accelerating
accelarator”

initiative,

http://www.gatesfoundation.org/GlobalHealth/Pri_Diseases/Tuberculosis/default.htm).

While

drug

discoveryfor

tuberculosis

(the

“TB

drug

acknowledging these important contributions, it must be questioned whether the isolated effort of
the Gates Foundation will be sufficient to promptly address such a broad and important publichealth problem. Much greater public leadership is needed.

35

Figure 4. Main players in the anti-TB drug discovery and development process

Time to sow new seeds now as all the low-hanging fruit have been eaten
The product-development partnership (PDP) model has considerably contributed to a burst ofactivities in R&D for TB and other neglected diseases (Moran et al., 2005), mainly by testing andreformulating existing drugs already used for other indications and pushing into pre-clinical andclinical development existing drug leads that would have been otherwise forgotten in laboratory
drawers for lack of an industry sponsor. Since improved therapies are urgently required thestrategy of adopting a development-oriented portfolio has probably been a sensible short-term
perspective. The critical question is whether this strategy will be successful in the long run. Thereare concerns that most of the “low-hanging fruit” have been already used up, and real
breakthroughs will require a strengthening of early-stage discovery research to identify newcompounds and targets. Without a thriving background of discovery-oriented translational research,
itself largely dependent on public funding, the PDP model is destined to fail in a longer-term
perspective. The renewed and welcome engagement of some pharmaceutical companies in TB
drug development is so far still too modest and risk (cost) averse to tackle this problem.
Another point that deserves attention is the lack of rational approaches in the discovery processof compounds currently being developed. Serendipity will only get us so far. In the market-driven

36

pharmaceutical sector, advances in genomic technologies, high-throughput screenings and X-ray
crystallography are facilitating both the understanding of infectious organisms and approaches torational drug design. These technologies need to be urgently and more comprehensively applied toneglected diseases if the pipeline for drug discovery and development is to remain full of realpromise. The reluctance of the pharmaceutical sector to invest in early-stage discovery research
for neglected diseases is leaving the pressing need to translate basic scientific knowledge intonovel targets and new therapies unmet. It is necessary to re-think the traditional roles played byacademia and pharmaceutical industry in drug discovery and development when it comes to drugs
for diseases like TB that do not represent an interesting market for the multinational
pharmaceutical industry. Without proper public sector engagement into translational research andimplementation of rational drug design, fast progresses will be severely hampered.

37

Appendix A: Promises from the basic research fieldIn 1979 Mitchison introduced the influential concept of distinct M. tuberculosis populations
characterized by distinct metabolic rate and therefore drug susceptibility, coexisting in an infectedindividual. Studies of surgically removed infected lung tissue clearly showed that infections inindividual patients are highly heterogeneous and comprise pathologically discrete lesions, including
open cavities, closed cavities and end-stage cavities (Kennedy et al., 1957; Kennedy et al., 1958;
Vandiviere et al., 1956). The only lesion in which abundant bacterial growth has been observed ison the inner surface of open cavities, a site that is insulated from the immune system. In thislocation, most bacteria are found inside macrophages (Kaplan et al., 2003) . Closed lesions
instead typically contained caseum, a wet/semi-dry chalky paste that results from tissue necrosis in
granulomas, and were not connected to the bronchi of the host. Clinical evidence supporting the
Mitchison hypothesis came from a study in which pulmonary lesions were resected from TB
patients after prolonged drug therapy (Vandiviere et al., 1956). Lesions classified as open andactive yielded colonies of predominantly drug-resistant tubercle bacilli in the normal timeframe (38weeks). In contrast, lesions that were closed and inactive gave colonies only with prolonged (310months) incubation. Surprisingly, the bacteria isolated form these lesions were almost uniformly
drug sensitive. Therefore, different types of lesions are present at the same time in an actively
infected TB patient, and can be seen as a wide range of distinct microenvironments that thebacteria have to adapt to in order to survive inside the host organism. Adaptation to those distinct
microenvironments most likely requires a fine-tuning of bacterial physiology and metabolism,
leading to metabolically distinct bacteria subpopulations co-existing in an actively infected patient.
Current multidrug therapy in TB patients leads to a relatively rapid sterilization of bacteria in the
sputum: typically sputum from most patients fails to yield bacteria in cultures within eight weeks(Garay, 2004). Considering that sputum-borne bacteria represent those that originate frommacrophages at the immediate cavity surface, the remaining 16 weeks of conventional therapy arelikely to be required primarily to eradicate bacteria from other types of lesions, possibly thoselocated in the necrotic or caseous regions that are remote from the cavity surface and are probably
characterized by a limited availability of oxygen and nutrients (Dannenberg and Rock, 1994;
Saunders et al., 1999). The microenvironment in which these treatment-refractory bacteria arefound in actively infected patients is potentially very similar to the microenvironment of the bacteria
in patients with latent disease.
It is therefore clear that a successful search for better, faster and cheaper drugs urgently
requires an improved understanding and consideration of the physiology and metabolism ofbacteria in vivo.
Although this idea is not new and pioneering studies aimed to highlight differences among invitro and in vivo metabolic strategies of M. tuberculosis were already performed in the 1950s(Bloch and Segal, 1956), significant advances have been made only recently thanks to the advent38

of molecular and genetics tools for mycobacteria and the availability of the full genome sequence(Cole et al., 1998). The application of genome-based biology, including array-based analysis ofmutant libraries and expression profiling, is allowing dissection of the fundamental genetics of M.
tuberculosis growth and persistence in animal models (Lamichhane et al., 2005; Sassetti and
Rubin, 2003; Shi et al., 2005); for review see (Manabe and Bishai, 2000; McKinney, 2000). Morerecently, crucial advances have been made in developing technologies that allow conditional genesilencing in M. tuberculosis (Blokpoel et al., 2005; Ehrt et al., 2005). The successful application ofthese systems for inducible gene silencing in vitro make the TB scientific community believe that it
will be finally possible to silence M. tuberculosis genes at specific stage of infection in animalmodels. This would allow investigators to turn a gene off at a particular time after infection has
begun, and thereby model the effect of treating the infection with an antibiotic directed against thegene product. Furthermore, the introduction of conditional gene inactivation will allow thecharacterization in vivo of genes that once inactivated preclude growth in vitro. Elucidation of thegenetic requirements for in vivo growth will not only lead to the identification of potential drug
targets that traditional screens would miss, but will also allow the refinement and improvement of invitro screens to mimic the environmental conditions that characterize in vivo growth.

Genes involved in energy metabolism and response to oxygen limitation
Oxygen limitation during granuloma development has been proposed to be one of the main
signals that result in a change in the metabolic state of bacteria. An in vitro model for the metabolic
alterations that are associated with oxygen depletion has shown that replication is dependent on
the presence of oxygen (Wayne and Hayes, 1996; Wayne and Sohaskey, 2001). In vivo, thenumber of bacilli in a lesion generally correlates well with the degree of oxygen (Canetti, 1955),
suggesting that oxygen supply might limit M. tuberculosis growth during infection. Moreover, when
an appropriate dose of tubercle bacilli is used to infect mice the resulting lung infection typicallyexhibit an acute phase (~20 days of exponential bacterial growth) and a subsequent chronic phaseduring which bacterial counts reach a plateau (~105-106 CFU/lung) (for a review see Orme andCollins, 1994). The arrest of bacterial growth during chronic phase is thought to be the result of theexpression of host adaptive T helper 1-mediated immunity and the consequent activation of
infected macrophages that produce inducible nitric oxide (NO) synthase which catalyses the
production of NO (for a review see (Flynn and Chan, 2001). NO acts as a potent oxidant andinhibitor of cellular respiration (MacMicking et al., 1997). Persistant bacilli are therefore thought tobe exposed in vivo to an environment which is poor in O2 and rich in NO (for a review see (Boshoffand Barry, 2005). Detailed analysis of the response of M. tuberculosis to hypoxia or exposure to
NO using microarray-based whole genome profiling has identified a set of genes that are rapidly
up-regulated in these conditions (Sherman et al., 2001; Voskuil et al., 2003). Among those genes
is the putative two-component regulatory system dosR-dosS/dosT (also called devR-devS,39

Rv3133c/Rv3132c). In bacteria two-component response regulator systems are an importantmeans by which a variety of environmental signals are transduced into a phenotypic response.
These systems typically consist of a membrane-bound sensor kinase and a soluble response
regulator that is activated by a histidine-aspartate phosphorelay to bind upstream of specific genes
and regulate their expression (Hoch and Silhavy, 1995). In the DosR-DosS/DosT two component
system dosS and dosT encode for sensor kinases while DosR encodes for a transcription factor. M.
tuberculosis mutants lacking DosS and DosT as well as the transcription factor DosR can nolonger activate DosR-dependent gene expression in response to reduced oxygen tension (Park etal., 2003; Roberts et al., 2004). In vitro data suggest that the components of the DosR-DosS/DosT
could be good targets to develop drugs against persister bacteria. However, currently available
data does not allow ruling out the precise role of the DosR-DosS/DosT in vivo. Indeed, while DosR
mutants are not attenuated for growth and survival in mouse tissue (Parish et al., 2003), DosR isrequired for virulence in guinea pigs (Malhotra et al., 2004). Future investigations should be aimed
at better assessing the role of these candidate drug targets in animal models.
In a recent study, transcription profiling of genes encoding components of the respiratory chain
and ATP synthesizing apparatus of M. tuberculosis was performed in vivo in infected mice (Shi etal., 2005). This study revealed that adaptation of M. tuberculosis to host immunity requires an
exchange in enzymes involved in respiratory pathways. While strong downregulation of protonpumping type I NADH dehydrogenase and aa3-type cytochrome c oxydase was observed during
chronic phase of infection, the less energy-efficient cytochrome bd oxidase and the nitratetransporter narK2 were clearly upregulated. These findings identify additional potential targets for
drugs directed against persisters bacilli and interest in this class of enzymes has been sparkled by
the discovery of efficient new antitubercular drugs that target respiratory components and ATP
synthesis (Andries et al., 2005). Validation of cytochrome bd oxidase or narK2 as potential drug
targets requires a detailed characterization of M. tuberculosis mutants lacking these enzymes andtheir ability to establish infection in vivo.

Genes encoding enzymes of the glyoxylate shunt
The strategy of survival of M. tuberculosis during chronic stages of infection is thought to involvea metabolic shift in the bacteria’s carbon source. Several lines of evidence suggest that pathogenic
mycobacteria primarily use fatty acid rather than carbohydrates as carbon substrate during
infection. Under these conditions, glycolysis is decreased and the glyoxylate shunt is significantly
upregulated (Wayne, 1994); (Gupta and Katoch, 1997). Isocitrate lyase (ICL) and malate synthase
are the two enzymes of this pathway, which has been described in various eubacteria, fungi, andplants. In Mycobacterium tuberculosis, isocitrate lyase activity is increased when the bacilli are in
an environment of low oxygen tension or in a transition from an actively replicating to a nonreplicating state (Kannan et al., 1985); (Wayne and Lin, 1982). ICL plays a crucial role for survival40

of M. tuberculosis bacteria in activated macrophages. It has also been shown that the geneencoding ICL (icl) is important for survival of M. tuberculosis in the lungs of mice during thepersistent phase of infection (2-16 weeks), but is not essential during the acute phase (0-2 weeks).
These findings emphasize the importance of this pathway to the bacteria in sustaining a chronic
infection (McKinney et al., 2000). A single malate synthase gene called glcB (also referred to as
aceB in M. tuberculosis CDC1551; MT1885; see www.tigr.org) has been identified in M.
tuberculosis encoding a malate synthase G (MSG) (Rv1837c; see (Cole et al., 1998). Since malate
synthase and ICL are part of the same metabolic pathway, inactivation of malate synthase is
expected to result in survival defects phenotypically similar to that observed in icl mutants.
Validation of malate synthase as a potential drug target awaits characterization of the in vivo
phenotype for bacteria lacking this enzyme. The crystal structure of malate synthase has beensolved and available data can facilitate development of specific inhibitors through structure-based
drug design (Smith et al., 2003)

Genes involved in the response to nutrients limitation
RelMtb. Intact granulomas formation are believed to be characterized by nutrient deprivation in
the form of amino acid and carbohydrate depletion in a process that is thought to be essential forcurtailing growth of microrganisms (Betts et al., 2002). When microorganisms encounter a nutrient
limited environment they slow down their growth rate dramatically and reduce levels of rRNA, tRNAand protein synthesis. Often RNA polymerase activities are modified, the activity of transportsystems is reduced and metabolism of carbohydrates, amino acids, and phospholipids isdecreased. Known as the stringent response, this broad alteration in metabolism is mediated by
the accumulation of hyperphosphorylated guanine nucleotides (p)ppGpp. The stringent response isreversed when environmental conditions become favourable and (p)ppGpp levels decrease.
Mycobacterium tuberculosis has one dual function enzyme, RelMtb, for the synthesis and hydrolysisof (p)ppGpp (Avarbock et al., 1999). In in vitro studies, relMtb mutants displayed a significant lower
aerobic growth rate than the wild-type bacterial strain. Moreover, long term survival of relMtb
mutants during in vitro starvation or nutrient run out in normal media was significantly impaired
compared to that of the wild-type strain (Primm et al., 2000). In mouse models of infection,

relMtbmutants presented normal initial bacterial growth but their ability to sustain chronic infection was
severely impaired (Dahl et al., 2003). More recently, relMtb was identified as one of the gene
required for short-term survival in mouse tissues in a microarray-based screen of M. tuberculosis
mutants suggesting that in addition to having a role in long-term persistence, relMtb may also be
important for short term survival in certain in vivo situations (Lamichhane et al., 2005).

41

Genes involved in cell wall and membrane metabolism
PcaA. Another mutant impaired in late-stage survival was discovered by Glickmann andcollaborators pcaA (Glickman et al., 2000), who identified a gene called pcaA encoding a
cyclopropane synthetase, a methyl transferase involved in the modification (cyclopropanation) of
the acyl chain of mycolic acids in mycobacterial cell wall. In mouse models, mutants lacking the
pcaA gene ( pcaA) showed no defects for early growth. However, at later stage of infection theability of the

pcaA mutants to maintain a stable peak was impaired. Although the effect of pcaA

deletion on bacterial persistence was subtle, inactivation of pcaA clearly decreased lung damage
and mortality from chronic M. tuberculosis infection. This suggests the possibility that
cyclopropanated mycolic acid can contribute to TB pathogenesis, either directly by inducing tissue
damage or indirectly by stimulating tissue-damaging immune responses. If this is true, drugs
targeting pcaA might offer the additional benefit of reducing tissue pathology.
At least two other cyclopropane synthetases (CmaA1, CmaA2) are involved in the site-specificmodification of mycolic acids (Glickman et al., 2001; Yuan et al., 1995). Crystal structure of these
enzymes has been determined and would be therefore available for structure-based inhibitor
design. The similarity of these enzymes in their active site suggests the possibility that one inhibitor
may be effective against multiple targets, reducing the potential for drug resistance (Huang et al.,2002). Whether the inactivation of the three genes encoding the cyclopropane synthetases(CmaA1, CmaA2 and pcaA) would more drastically affect bacterial persistence and pathogenesis
in animal models remains an open question that deserves further investigation.

PE-PGRS. The PE-PGRS protein family counts approximately 60 members and is unique tomycobacteria. The function of PE-PGRS family members is unknown. There is evidence that someare cell-wall associated and may play a role in modifying host immune responses (Banu et al.,
2002; Brennan et al., 2001). Mutation of two PE-PGRS genes in M.marinum produced strains
unable to replicate in macrophages and with impared persistence in granulomas (Ramakrishnan etal., 2000). A recent study using transposon mutagenesis identified the PE-PGRS as a familyenriched in essential genes (Lamichhane et al., 2003). Although some members of the PE-PGRS
family present interesting features, further investigations about the role of PE-PGRS proteinsduring infection in mouse models is required before they can be taken in consideration as potential
drug targets. Moreover, development of drugs targeting PE-PGRS proteins might be complicated
due to the redundancy existing among family members.

MmpL. Members of the mycobacterial membrane large protein family (MmpL) are involved in
the transport of a range of substrates across the membrane. The first member of this protein family
that was found to play a role in M. tuberculosis survival in vivo is MmpL7. M. tuberculosis mutants
lacking MmpL7 activity are severely attenuated for growth in the lungs but not in the spleen and42

liver of animal models (Camacho et al., 2001; Cox et al., 1999). Several independent studies haveidentified the MmpL7 gene as part of the M. tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus
that plays a major role in biosynthesis and secretion of phtiocerol dimycocerosate (PDIM), a cellwall associated lipid necessary for M. tuberculosis virulence (Camacho et al., 1999; Cox et al.,
1999; Pinto et al., 2004). In particular, PDIM seems to have a role in protecting M. tuberculosis
bacteria from the toxic action of nitric oxide released by activated macrophages (Rousseau et al.,2004).
Recent studies have showed that, with the exception of mmpL3 that is apparently essential for
viability, members of the MmpL protein family are not required for growth in vitro but they seem toplay a crucial role for survival of M. tuberculosis in vivo in mouse models (Domenech et al., 2005;
Lamichhane et al., 2005). When assessed for growth kinetics and lethality in a murine model oftuberculosis, mutants with inactivation in mmpL8 and MmpL 11 were able to establish a normal
infection but they were significantly attenuated for lethality in time-to-death studies. On the other
hand, bacterial strains carrying mutation in MmpL4 and MmpL7 were found to have impaired
growth kinetics and impaired lethality (Domenech et al., 2005). In another independent study,
preliminary results showed that mutants with disruption in mmpL4, mmpL5, mmpL7, mmpL8,
mmpL10, mmpL11 showed significant attenuation for growth in the lungs, while only mmpL4
showed a survival defects in the spleen (Lamichhane et al., 2005). Further investigation should aim
to identify the substrates that are transported across the membrane by the MmpL family members.
In particular it would be interesting to identify MmpL4’s substrates, since bacteria lacking MmpL4
showed impaired survival in lungs and spleen in mouse models. Thus, drugs targeting MmpL
proteins or enzymes involved in the biosynthesis of the substrates transported by MmpL proteins
across the membrane could effectively impair in vivo survival of M. tuberculosis.

Genes involved in transcriptional regulation.
Sig F. RNA polymerase-factors are transcription factors that are upregulated under specificconditions and direct the transcription machinery to a specific group of genes necessary for thesurvival of the bacterium under those environmental conditions. One of these-factors, Sig F, isexpressed during stationary phase, when bacterial growth and division are slowed, and is virtually
undetectable when cells are dividing rapidly (DeMaio et al., 1996). A strain of M. tuberculosis inwhich the gene SigF was inactivated has been constructed and tested in mouse models for TB
infection. While loss of SigF did not prevent the mutant strain from producing lethal infection, death
was significantly delayed in mice infected with the mutant strain. More interestingly, in vitro
experiments showed that SigF mutants were more susceptible to rifamycin drugs (Chen et al.,2000). It is interesting to speculate that SigF could be taken in consideration as a target fordevelopment of a “drug-enhancing drug” that would not have intrinsic bactericidal activity and yet it
would accelerate treatment by promoting the action of conventional drugs (McKinney, 2000).

43

Genes involved in promoting M. tuberculosis survival inside macrophages.
M. tuberculosis is an obligate pathogen whose primary target cells are macrophages. The
successful parasitization of macrophages by pathogenic mycobacteria involves the inhibition ofseveral host-cell processes. Host processes inhibited by pathogenic mycobacteria include thefusion of phagosomes with lysosomes, antigen presentation, apoptosis and the stimulation ofbactericidal responses due to the activation of pathways involving mitogen-activated protein
kinases (MAPKs), interferon- (IFN- ) and calcium (Ca2+) signalling (for a review see (Koul et al.,
2004; Nguyen and Pieters, 2005).

Inhibition of phagosome maturation
One of the strategies by which pathogenic mycobacteria survive the bactericidal mileu ofmacrophages is the inhibition of fusion of their intracellular niche (the phagosome) with lateendosomes and lysosomes ((Armstrong and Hart, 1971; Russell, 2001). Phagosomes are vesicles
that are formed by invagination of the plasma membrane during endocytosis and subsequently
fuse with primary lysosomes to degrade engulfed material, a processes also termed as“phagosome maturation”. Lysosomes are membrane-limited cellular organelles with a low internal
pH and contain enzymes for degradation of polymers such as proteins, DNA, RNA polysaccharides
and lipids. Normally, phagocytosed microorganisms are rapidly transferred from phagosomes tolysosomes and are then digested and degraded by hydrolytic enzymes. However, pathogenic
mycobacteria are directed to phagosomes that subsequently fail to fuse to lysosomes (Armstrongand Hart, 1975).
The M. tuberculosis cell wall component phospahatidylinositol-3-phosphate (PI3P) analog
glycosylated lipoarabinomannan (Man-LAM) has been reported to block phagosome maturation by
inhibiting a signalling cascade that consist of Ca2+, calmodulin and phospahatidylinositol-3-kinase(PI3K) (Fratti et al., 2003; Rojas et al., 2000). The arrest of phagosome maturation by Man-LAM
represents an effective mechanism that is used by mycobacteria for long term survival in host cell.
In view of the central role played by Man-LAM in mediating intracellular survival of M. tuberculosis,
genes involved in the biosynthetic pathway of Man-LAM represent potential targets for novel antiTB drug.
In addition to cell wall lipids, the serine/threonine protein kinase G (PknG) was found to play arole in the arrest of phagosome maturation (Pethe et al., 2004; Walburger et al., 2004). When thegene encoding PknG was disrupted in M. bovis BCG, the resulting mutant mycobacterial strain
was immediately transferred to lysosomes and was not able to survive inside macrophages(Walburger et al., 2004). The ability of PknG to block lysosomal delivery suggests that this kinasemight be valuable target in the development of drugs that could induce mycobacterial death inside
the macrophages. A screen for PknG inhibitors identified a tetrahydrobenzothipene that specifically44

inhibits the PknG kinase activity. When added to infected macrophages, this compound induces
the fusion of phagosomes to lysosomes and mediates killing of mycobacteria inside macrophages(Walburger et al., 2004). Preliminary subcellular localization studies suggest that PknG is secreted
into the phagosomal lumen and cytosol of infected macrophages. If this is proven to be the case it
would represent a crucial advantage in terms of drug delivery. By targeting a secreted molecule
such as PknG indeed, transport of anti-microbial agents through the extremely impermeable
mycobacterial cell wall can be circumvented, greatly improving the accessibility of the compounds
to their target. Further investigations should be aimed to confirm the role of PknG in M.
tuberculosis species. Validation of PknG as a potential drug target requires analysis of M.
tuberculosis mutants lacking PknG and assessment of their ability to survive and successfully
establish infection in vivo.

Resistance to nitric oxide stress
It has been showed that production of reactive nitrogen intermediates by induction of nitric
oxyde synthase (iNOS) in activated macrophages is necessary to protect against tuberculosis(Chan et al., 1992). Macrophages activation in the response to M. tubeculosis is mainly mediatedby IFN- . Consistently, mice deficient in the gene for IFN-

or in the gene for iNOS succumb

rapidly to M. tuberculosis (Dalton et al., 1993; Flynn et al., 1993; MacMicking et al., 1997). A recent
study identified the genes that are required by M. tuberculosis to resist to the harmful effect ofreactive nitrogen intermediates (Darwin et al., 2003). These genes encode for the subunits of the
mycobacterial proteasome. The proteasome is a multisubunit molecular machine that is highly
conserved from archibacteria to humans and is responsible for the proteolysis of cellular proteins.
M. tuberculosis has adapted the proteasome machinery to protect itself from the killing effect ofnitric oxide and deletion of genes that encode proteins involved in the formation of proteasome
causes hypersensitivity of the bacilli to nitric oxide (Darwin et al., 2003; Pieters and Ploegh, 2003).
In an independent study, a comparison of the intraphagosomal gene expression profile of M.
tuberculosis in both resting and activated macrophages identified several genes that are involved
in induction of persistence, fatty-acid metabolism and resistance to nitric oxide (Schnappinger et al.,2003). Further characterization of these gene products will provide information about the survival
strategies of this pathogen and also help to identify new targets.

45

Appendix B: Update on compounds in the pipeline
By the time this report has reached its final format, few new compounds have entered the pipeline
under the discovery phase. Hereafter we briefly review the compounds for which information were
publicly available.

Malate Synthase Inhibitors (GSK, Rockefeller University, Texas A&M)
As the isocitrate lyase (ICL), the malate synthase is an enzyme of the glyoxylate shunt, a metabolic
pathway of M. tuberculosis that appears to be upregulated during the chronic stage of infection.
Since malate synthase and ICL are part of the same metabolic pathway, inactivation of malate
synthase is expected to result in survival defects phenotypically similar to that observed in icl
mutants (see Appendix A). Identification of inhibitors for ICL has proven lengthy and laborious dueto the conformation of the enzyme active side (see paragraph 3.1). Therefore, efforts are currently
being focused on identifying inhibitors of the second identified component of the glyoxylate shunt,the malate synthase.

Riminophenazines (Institutes of Materia Medica/BRTI)
Riminophenazines were specifically developed as drugs active against M. tuberculosis but thesecompounds are also active against many other mycobacterial infections, particularly those causedby M. leprae and the M. avium complex (MAC). Clofazimine, is the lead compound in this series.
With the introduction of rifampicin and later pyrazinamide for the treatment of tuberculosis, interestin riminophenazines abated, mainly because the drug is not as powerful as either rifampicin or
isoniazid (Reddy et al., 1999). With emergence of multidrug-resistant (MDR) tuberculosis, theinterest in riminophenzines was renewed. Clofazimine has indeed showed activity against MDR
strains of M. tuberculosis in animal models (Klemens et al., 1993). This drug is currently included in
the

list

of

the

second

line

drugs

recomended

by

WHO

(http://whqlibdoc.who.int/publications/2006/9241546956_eng.pdf).
Investigation of new analogues of clofazimine could lead to the identification of compounds that
increased activity and cause less pigmentation of internal organs and fatty tissues (Jagannath etal., 1995)
Capuramycins (Sankyo/Sequella)
Capuramycin analogues have been shown to have selective antibacterial activity against
mycobacteria (Muramatsu et al., 2003). These compounds inhibit the phospho-N-acetylmuramylpentapeptide translocase and therefore interfere with the biosynthesis of mycobacterial the cellwall. When tested against M. tuberculosis, Capuramycins analogues appear to be equally active
against drug-susceptible and drug resistant strains. However, the MIC range showed by these46

compounds was significantly higher than the MIC for rifampicin or isoniazid when tested against
drug-susceptible strains (Koga et al., 2004). Further studies are required to thoroughly assess theactivity of the capuramycins analogues in vivo

Proteasome Inhibitors (Cornell University)
As described in Appendix A, the proteasome (i.e. the protein degradation machinery of the cell)
represents an interesting potential new target for anti-tuberculosis drugs. The activity of M.
tuberculosis proteasome, appears to be important for protecting the bacteria from the killing effectof the nitric oxide produced in activated macrophages. deletion of genes that encode proteins
involved in the formation of proteasome causes hypersensitivity of the bacilli to nitric oxide. Drugs
targeting the proteasome are expected to be active against MDR M. tuberculosis strains as theywould act through a completely novel mechanism.

Protease Inhibitors (Medivir)
Proteases are a special class of proteins, operating as extremely precise biological 'scissors' cutting long protein chains, thereby increasing or decreasing a particular protein's activity.
Since 2001, Medivir has been in collaboration with scientists at Bart's, and the London School of
Medicine, Queen Mary College, the University of London in the development of new antibiotics-anarrangement funded by the UK Department of Trade and Industry (in its Applied Genomics
initiative), the UK's Medical Research Council and Medivir.
This project is founded on the expertise within Queen Mary Hospital in identifying and validating
bacterial proteases as target enzymes for pharmaceuticals. Medivir is using its pharmaceuticals
knowledge and technology platforms in the protease sphere to develop new antibiotics. As part ofthis project, the genome of M. tuberculosis is being examined to identify genes encoding forproteases of interest as drug targets, to the purpose of developping protease inhibitors as
antibiotics

againstthesebacteria(http://www.medivir.se/v2/eng/news/show-

news.asp?trigYear=2001&id=29).

The following drug discovery projects are being carried out in collaboration with the TB Alliance.

Bifunctional Molecules (Cumbre- TB Alliance)
Cumbre Pharmaceuticals Inc and the TB Alliance have just launched a joint program to develop
new anti-tuberculosis agents.
Under the terms of the agreement, the parties will work on the design, synthesis and optimizationof two different classes of multi-functional antibiotics. The collaboration will utilize Cumbre's uniqueantibiotic discovery and development technical platform to identify and deliver novel antibiotic
compounds. The TB Alliance will have exclusive rights to these compounds for the treatment of47

tuberculosis and other neglected diseases, while Cumbre will retain the rights to pursue
compounds

for

use

in

other

infectious

diseaseareas(http://www.tballiance.org/7_1_1GenericNewsArticles.asp?itemId=728).

Bacterial Topoisomerase Inhibitors (GlaxoSmithKline-TB Alliance)
The DNA Topoisomerases are enzymes that control DNA topology and are vital for cellular
processes that involve duplex DNA, namely replication, recombination and transcription. DNA
gyrase, the single type II topoisomerase of M. tuberculosis, is the molecular target offluoroquinolones. Due to limited public information about this project, it is not known whether the
new compounds under development will target DNA gyrase or DNA topoisomerase I

48

Appendix C: Extensive Drug Resistant Tuberculosis (XDR-TB)
Multi-drug resistant tuberculosis (MDR-TB) is a form of tuberculosis resistant to at least the two
principal first-line drugs rifampicin and isoniazid. The Global XDR-TB Taskforce convened by theWorld Health Organization in October 2006 defined extensive drug-resistant tuberculosis (XDRTB) as a form of tuberculosis resistant not only to rifampicin and isoniazid, but also to certain
second-line drugs (at least one fluoroquinolone and one of the three injectable drugs kanamycin,
amikacin or capreomycin).

XDR-TB in itself is not a new problem. The existence of XDR-TB strains has been known topractitioners working in Eastern European and Central Asian countries. What is alarming about the
recent outbreak is that it is occurring in a country with very high HIV prevalence (South Africa), andrisks spreading extremely rapidly amongst HIV positive people.

XDR-TB in KwaZulu Natal, South Africa – the spread of a new strain
A survey among suspected TB patients in the rural district of KwaZulu Natal between January2005 and March 2006 revealed that 221 (41%) of 544 patients that tested culture positive forM.tuberculosis were infected by multi-drug resistant strains.

53 patients out of 221 (24% of MDR or 10% of all culture positive patients) were infected with XDRstrains. 51% of the XDR patients had no prior TB treatment, suggesting that they had been newly
infected by XDR-TB strains, and that resistance did not develop during treatment.

52 of the 53 XDR-TB patients died. The combination of XDR-TB and HIV infection leads patientsto develop a highly aggressive form of tuberculosis that causes death in a very short time.

The emergence and rapid spread of XDR-TB in high HIV prevalence settings represent a major
threat to global health.

The phenomenon is a demonstration of the limitations of TB control

programmes, which have been relying on outdated tools for TB diagnosis and treatment.

XDR’s implications for the TB drug and diagnostics R&D pipelines
The immediate responses of the public health community must not focus solely on strengthening
control programmes. It is also urgent to mobilise all necessary resources for the rapid delivery of
new drugs and diagnostic tools.

49Concerning drugs, it is crucial that the pipeline be filled with compounds that act through novel
mechanisms which are able to target novel molecular targets, in order to avoid cross-resistancewith drugs currently in use.

Currently, there are a few new promising candidate drugs in the clinical phase of development.
These candidate drugs have been shown to be active against MDR-TB strains in vitro

andtherefore have the potential to be effective against MDR-TB in human patients.

There is an urgent need for innovative thinking in the field of clinical trials for new TB drugs, inorder to speed up the development of these new drugs and accelerate their delivery to patients.

A major limitation currently is the difficulty of diagnosing patients with TB. This problem is evenmore acute in the case of XDR-TB because the disease is so rapidly fatal that most patients will
die before the results of their diagnosis are available. Rapid, reliable and field adapted diagnostic
tools for TB and drug resistant forms of TB are an integral part of treatment strategies and urgently
need to be developed.

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