92 Infectious Disorders - Drug Targets 2007, 7, 92-104

Programs to Facilitate Tuberculosis Drug Discovery: The Tuberculosis Antimicrobial

Acquisition and Coordinating Facility

R. C. Goldman*1, B.E. Laughon1, R. C. Reynolds2, J. A. Secrist III2, J. A. Maddry2, M.-A. Guié2, A. C.

Poffenberger2, C. A. Kwong2 and S. Ananthan2

1
Department of Health and Human Services, National Institute of Allergy and Infectious Diseases, Division of AIDS, Therapeutics
Research Program, Complications and Coinfections Research Branch, Bethesda, Maryland 20892, 2Southern Research Institute, P.O.
Box 55305, Birmingham, Alabama 35225-5305

Abstract: There is a real need to discover new drugs that are active on drug-resistant tuberculosis (TB), and for drugs that will shorten
the time of therapy. Large pharmaceutical companies have traditionally led the quest for discovering and developing new antiinfective
agents but this is not the case when it comes to diseases like tuberculosis that primarily occur in resource restricted countries. Throughout
the world many research groups are actively engaged in the scientific discovery of new TB drugs. Unfortunately, most research
laboratories do not have the necessary safety facilities or resources for all facets of TB drug discovery. The Tuberculosis Antimicrobial
Acquisition and Coordinating Facility (TAACF) was established in order to make comprehensive testing services available at no cost to
research laboratories with an interest in discovering new TB drugs. The TAACF is a consortium of contracts managed and funded by the
National Institute of Allergy and Infectious Diseases (National Institutes of Health, Bethesda, MD) as a resource to support preclinical
drug discovery and development. The core of the TAACF is the Southern Research Institute, Birmingham, AL, which supports
compound acquisition, storage, medicinal chemistry, and high throughput assays. Other collaborating groups provide biological data on
antimycobacterial activity and cytotoxicity, preliminary in vivo toxicity, oral bioavailability and efficacy in animal models, specialty
testing (such as activity against non-replicating persistent bacteria), and assistance in technology transfer for developing comprehensive
promotional packages and facilitating partnerships with pharmaceutical companies for drug development. The TAACF program and
recent progress that has been publicly disclosed by suppliers is reviewed. There are many aspects promising of the program that will not
be discussed due to confidentially.

Keywords: Tuberculosis, drug discovery, Mycobacterium, screening.

INTRODUCTION Mr. Badman: Presented to the World in a Familiar Dialogue

Tuberculosis has afflicted thousands of years of morbidity and
mortality upon humankind. Throughout history tuberculosis was
called by many names, sometimes in relation to the tissue being
infected (e.g. Potts’s disease or tuberculous spondylitis, as
described by Percival Pott, a London surgeon, 1714 to 1788). The
oldest reports of TB date back to the Predynastic time in Egypt,
where the presence of Pott’s disease was diagnosed based on an
archeological finding of pathology in thoracic vertebrae. A report
by Sir Marc Armand Ruffer, Professor of Bacteriology at Cairo
Medical School in the early 1900's, described the collapse of
thoracic vertebrae producing the characteristic angular kyphosis
(hump-back). More modern methods confirmed Dr. Ruffer’s
findings. Microbial DNA indicative of Mycobacterium tuberculosis
(Mtb) was isolated and sequenced from a 5,400-year-old
Predynastic Egyptian skeleton that exhibited a spinal deformity
consistent with Pott's disease [1], and such studies were confirmed
by more extensive molecular analysis [2,3,4].
The symptoms of tuberculosis disease were familiar to
Hippocrates (460-377 B.C.E.) who described it as "consumption"
because the characteristics seen in late stages appears to consume
patients. In the early twentieth century, TB was commonly called
"consumption" or "white plague". Tuberculosis was also rather
fittingly referred to as "the captain of all these men of death" by
John Bunyan (1628 to 1688) in his writing ‘The Life and Death of
1
Between Mr. Wiseman and Mr. Attentive” .
The defining point in TB disease was the identification of the
causative agent by Robert Koch, the German biologist in 1882. He
is quoted as stating "If the number of victims which a disease
claims is the measure of its significance, then all diseases … must
rank far behind tuberculosis." However, it took another sixty years
before streptomycin was discovered as a miracle drug for treating
TB. This excitement was short-lived as resistant strains readily
appeared during mono-therapy. As additional drugs came into
clinical use, e.g., aminosalicylic acid followed by isoniazid (INH) a
few years later, combination therapies were initiated to prevent
resistance selection. All of this took place during the period when
chemotherapy for infectious disease was rapidly evolving and
leading to the discovery and development of the many successful
treatments discovered during the second half of the twentieth
century. However, there were two problems: 1) bacteria become
resistant to antibiotics, and 2) improper administration of antibiotics
fueled the development and spread of resistant bacteria, including
Mtb. Statistics at the turn of the 21st century were not encouraging.
Worldwide estimates for new cases of MDR-TB (defined as
tuberculosis strains resistant to at least INH and rifampin) increased
from 272,906 (95% confidence interval [CI], 184,948–414,295)
worldwide in 2000 to 424,203 (95% CI, 376,019–620,061), or 4.3%
(95% CI, 3.8%–6.1%) of all new and previously treated TB cases
[5]. Mtb continued to evolve, and given the increased complexities
in procuring and delivering therapy for MDR-TB, we are now faced
with global outbreaks of XDR-TB, TB resistant to INH, rifampin,
any fluoroquinolone and at least one of three injectable second-line

*Address correspondence to this author at the Complications and
Coinfections Research Branch, Therapeutics Research Program, DHHS/
NIH/NIAID/Division of AIDS, 6700B Rockledge Drive, Room 5226, MSC
7624, Bethesda, MD 20892, Courier/express mail ZIP 20817; Tel: 301-496-
8424; Fax: 301-451-5481; E-mail: rgoldman@niaid.nih.gov
1
In this dialog Mr. Attentive asks “Pray of what disease did Mr. Badman die, for now I
perceive we are come up to his death? Mr. Wiseman replies, “I cannot so properly say
that he died of one disease, for there were many that had consented, and laid their
heads together to bring him to his end. He was dropsical, he was consumptive, he was
surfeited, was gouty, and, as some say, he had a tang of the Pox in his bowels. Yet the
Captain of all these men of death that came against him to take him away, was the
Consumption, for 'twas that that brought him down to the grave”.

1871-5265/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

Programs to Facilitate Tuberculosis Drug Discovery
drugs (i.e., amikacin, kanamycin, or capreomycin). Please see the
separate review on XDR-TB elsewhere in this issue of Infectious
Disorders, Drug Targets. Moving forward there are many
challenges to effectively managing the global TB effort. We need to
more effectively deliver treatments for drug-sensitive TB in order to
stop the selection of additional resistant strains. Areas where MDR-
TB is already established need to effectively deliver the more
complex therapeutic regimens for MDR-TB to limit spread and to
limit selection for further resistance on the path to XDR-TB.
Procedures must be adopted to contain and limit the spread of
XDR-TB. Finally, new tools are needed to effectively diagnose and
deliver appropriate treatment and prevention of TB using existing,
and new drugs as they emerge for treating sensitive or drug-
resistant TB.
THE SEARCH FOR NEW DRUGS
The search for new drugs to effectively treat TB, including
drug-resistant forms, has made significant progress with four new
drugs in clinical trials (see Laurenzi et al. elsewhere in this issue).
Unfortunately, none of these agents will be available for approved
clinical use for several years, and there is always the specter of
clinical failure. In addition, the superior activity of elevated doses
of rifapentine combined with moxifloxacin (MFX) [6,7] has led to
plans for a four year clinical trial of intermittent therapy with a high
dose of rifapentine combined with MFX in Mozambique, Zambia,
Zimbabwe and South Africa (http://www.medicalnewstoday.com/
medicalnews.php?newsid=63306). The hope is that the new
regimen termed ‘RIFAQUIN’, will shorten the treatment time from
six to four months.
TB can be controlled and theoretically eliminated, as was done
for smallpox and nearly accomplished for polio. Countries with
well-established, modern healthcare systems have demonstrated
that with the required facilities and resources, TB can be effectively
controlled. However, regions lacking the required health care
infrastructure for delivering appropriate TB therapy will continue to
experience increases in the incidence of TB and especially drug-
resistant TB. The continued evolution and selection of drug-
resistant TB, including MDR-TB and XDR-TB, will add to future
challenges in controlling global TB.
Unfortunately, in recent years the larger multinational drug
companies have not expended significant resources to discover and
develop new drugs for TB. It is understandable that the industry is
reluctant to invest heavily in TB drug discovery and development,
given the high cost of bringing a new drug to market (approaching
one billion dollars US by some estimates) and the poor economic
conditions in countries with high incidences of TB disease. One
only need look at the issues developing around HIV drugs,
including demands for lower prices and the recent announcement
from Thailand that it would apply the World Trade Organization's
agreements on intellectual property that allows a government to
issue a compulsory license allowing the manufacture, import and
sale of generic versions of drugs in case of a national public health
emergency. In this case both HIV and cardiovascular medications
were targeted. This problem represents another truth that global
health care is struggling with: discovering and developing new
drugs is very expensive and the companies that do so must be able
to recoup investment and development costs or they will no longer
be viable commercial entities. If the private sector, which has been
very successful in the past in delivering new medicines, can no
longer recoup costs then the public sector will be required to take
on this task. Regardless, the costs will not go down and ultimately
someone will have to pay.
Another issue unique to TB drug discovery is the fact that many
researchers do not have the appropriate facilities to conduct TB
research. The National Institute Allergy and Infectious Diseases
(NIAID) established the Tuberculosis Antimicrobial Acquisition
and Coordinating Facility (the TAACF) in order to provide access
Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2 93
to new drug testing facilities. This program allows investigators,
predominantly chemists, with an interest in discovering new TB
drugs to have compounds tested at no cost, with retention of
supplier intellectual properties rights. The extensive use of the
TAACF provides an indication of how many laboratories with an
interest in TB drug discovery did not have the appropriate facilities
to conduct much of the required testing (e.g. BSL2 and 3 laboratory
and animal care space). This fact was documented by a survey of
TAACF providers which revealed that most providers did not have
access to many of the needed resources (Table 1).
Table 1. Percent of TAACF Suppliers without Access to
Specific Testing
Percent without
Capable of Performing Tests
Access
MIC 70
Activity against Mtb in macrophages 90
Cytoxicity of lead compounds to mammalian cells 76
In vivo efficacy testing in animal models 85
High Throughput Screening (cells and enzyme
85
targets)
Activity against ‘latent’ TB (i.e. adapted to low
90
oxygen)
Pharmacokinetics analysis of lead compounds in
93
animals
TB THERAPIES
The standard regimen recommended by the WHO for treating
drug-susceptible TB consists of an initial phase of therapy with two
months of INH, rifampicin, pyrazinamide and ethambutol. The use
of ethambutol can be omitted when treating HIV negative adults
and children with negative sputum smears who do not have
extensive pulmonary tuberculosis or severe forms of extrapul-
monary disease. The preferred continuation phase consists of INH
and rifampicin given for four months. INH and ethambutol given
for six months is an alternative continuation phase regimen but is
associated with a higher rate of failure and relapse, especially in
patients with HIV infection. WHO guidelines recommend specific
treatments for MDR-TB and pan-resistant TB, consisting of various
combinations of first and second-line TB drugs (see Laurenzi et al.
in this issue). Effective treatment of MDR TB requires adminis-
tration, for 18-24 months, of four to six drugs to which the infecting
bacilli are susceptible, including multiple second-line drugs.
The NIAID TB research program grew out of the MDR-TB
crisis in New York City in the 1990s. Using emergency appro-
priations, the NIH and CDC mobilized resources to rapidly rebuild
the TB research infrastructure and put into place product develop-
ment activities to prime the pipeline for development of new drugs,
vaccines, and clinical research capabilities. Dr. Scott Franzblau’s
development of a rapid assay based on the dye Alamar Blue [8] led
to the establishment of a 96-well based assay to detect inhibition of
growth of Mtb and a VERO-cell assay for drug cytotoxicity. This
system became standardized and validated for drug testing at the
National Hansen’s Disease Center in Carville, LA, through an
interagency research agreement with NIAID. Over the last decade
and a half, the TAACF contracts have assisted research chemists
from every continent on the globe and screened over 90,000 unique
compounds. From this effort have emerged a dozen lead com-
94 Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2
pounds, over 200 publications, more than 20 NIH research grants
generating data from these resources, and contributions to the
advancement of four new candidate drugs in advanced preclinical
or early Phase I and II clinical trials. However, attrition rates for
new chemical entities in drug development remains high and
without the serious commitment of the large pharmaceutical
companies, the need for continued assessment of compounds by
government-sponsored programs continues. In this review we will
summarize many of the accomplishments of the TAACF but this
will necessarily be limited to data that has been publicly disclosed
by suppliers; proprietary work in progress on lead optimization and
advance animal studies will not be discussed.
OPERATION OF THE TAACF
Southern Research Institute (SRI) continues, as it has for over
12 years, to serve as the functional hub of the TAACF program [9].
During these years SRI provided support in the form of medicinal
chemistry, compound acquisition, sample repository, and data
management services. Recently, SRI’s role was expanded with a
new role as the program’s principal provider of in vitro testing
services.
SRI’s contributions are based on it’s over sixty-year history in
drug discovery. SRI scientists identify potential sources of suitable
test compounds, execute legal screening agreements with candidate
participants, acquire and prioritize compounds for screening against
Mtb, maintain a computerized inventory for tracking compounds
and data, and coordinate and distribute compounds for evaluation.
SRI has approved and processed nearly 90,000 compounds through
agreements with both academic institutions and the pharmaceutical
industry worldwide, and has identified a number of exciting new
leads. Thus, focusing the combined resources of industry, acade-
mia, and NIAID on the TB problem has expedited the discovery of
new lead compounds active against Mtb.
EVOLUTION OF TECHNOLOGIES
The adoption of new and evolving technologies has been
essential to the long-term success of the TAACF. For example, at
the program’s inception, potential suppliers were identified and
their names and contact data stored in a spreadsheet. As the number
of suppliers increased, SRI researchers built, and continue to
maintain, an exhaustive relational database that now contains over
10,000 individual researchers, compound suppliers and potential
suppliers. Other functional areas of the program have benefited
from evolving technological advancements. For instance, the
quality and number of chemical database software products has
grown enormously since the mid-1990s, and SRI rapidly moved to
adopt new technologies that help to automate the importation,
storage, and accession of chemical structure and bioassay results.
Early on, the cheminformatics team used stand-alone desktop
databases (ISIS/Base from MDL) but within five project years (the
late 1990s) recognized the need for, and began exploring, more
robust, reliable, and secure client/server solutions. SRI now uses the
ActivityBase suite (ID-BS), which by design allows for broad
customizations and is itself an ever-evolving technology. Software
for evaluating structure activity relationships (SAR) has improved
substantially and SRI routinely employs such programs as Sybil
Distill to cluster actives.
Acquisition and screening of candidate compounds have
evolved as a result of technological advancements, experience, and
ingenuity. Medicinal chemists who review candidate compounds
increasingly apply stringent criteria in the selection of new
compounds submissions. For example, SAR from previous testing
can preclude acceptance of certain chemical classes. In terms of
screening, significant improvements have been realized. Initially,
screening occurred at a remote laboratory using the more labor-
intensive methodology, the BACTEC 460 radiometric assay. That
assay was replaced fairly early in the program (~1997) with a more
Goldman et al.
time- cost-effective and less labor-intensive technique utilizing the
fluorescent dye Alamar Blue [10]. Recently, the TAACF imple-
mented high throughput screening technology in 384-well format
for fast and reliable primary screening with minimum amount of
sample. More detailed descriptions of current approaches to
TAACF compound acquisition and screening are in the sections
following.
COMPOUND ACQUISITION AND PRIORITIZATION
Compound acquisition is one of the key activities of the
TAACF program, and involves identification of unique, medi-
cinally relevant samples that have or are likely to show biological
activity, particularly antimicrobial activity. A major thrust of the
TAACF program is to acquire and screen samples in chemical
classes unique from the known, currently used antibacterial and
antitubercular agents. By doing so, the chances increase for
discovering agents with new mechanisms of action that might show
activity against multidrug or extremely drug resistant (MDR and
XDR tuberculosis, respectively) forms of tuberculosis. Medicinal
chemists at SRI review the relevant literature and work with
biologists and chemists in the areas of antimicrobial and antimy-
cobacterial target identification, drug design and synthesis to select
new candidate compounds for the TAACF program. Candidate
compounds have been identified from a wide variety of sources
throughout the world, and they include drug-like molecules from
many diverse chemical classes.
Next, after obtaining approval from the Project Officer,
medicinal chemists contact prospective suppliers and initiate a
Material Evaluation Agreement to cover TAACF testing. While the
majority of acquired samples are synthetic compounds or libraries
of small molecules, the acquisition of pure natural compounds is
encouraged since many known antibacterial drugs are of natural
origin. The structures of all compounds are verified prior to
acceptance as new to the NIAID tuberculosis chemical database. In
addition to sample requirements for initial in vitro screening (1.0
mg to 10 mg depending on submission format and extent of initial
testing), compound suppliers are asked to commit to providing
sufficient amounts of compound for advanced in vivo testing when
warranted by in vitro test data.
COMPOUND TESTING
Until September 2006, the National Hansen's Disease Programs
(NHDP) TAACF facilities, now located in Baton Rouge, LA and
led by Dr. Jim Krahenbuhl, performed in vitro screens for the
TAACF program. NHDP received compounds for multiple assays,
as outlined below. At the completion of each assay, results are
validated and data delivered to the compound supplier.
The Level 1 screen is conducted at 6.25
g/ml (or molar
equivalent of highest molecular weight compound in a series of
congeners) against Mtb H37Rv (ATCC 27294) in BACTEC 12B
medium using the Microplate Alamar Blue Assay (MABA) [10].
Compounds exhibiting fluorescence are tested in the BACTEC 460-
radiometric system. Compounds giving <90% growth inhibition in
the primary screen (MIC >6.25
g/ml) are not generally evaluated
further.
The Level 2 screen consists of two assays: 1) determination of
minimum inhibitory concentration (MIC) and 2) determination of
50% inhibitory concentrations (IC50) on VERO cells. Compounds
demonstrating at least 90% inhibition in the Level 1 screen are re-
tested at lower concentrations against M. tuberculosis H37Rv to
determine the actual MIC in the MABA. The MIC is defined as the
lowest concentration effecting a reduction in fluorescence of 90%
relative to controls. Concurrent with the determination of MICs,
compounds are tested for cytotoxicity (IC50) in VERO cells at
concentrations less than or equal to 62.5
g/ml or 10 times the MIC
for M. tuberculosis H37Rv. After 72 hours exposure, viability is
assessed on the basis of cellular conversion of MTT into a formazan
Programs to Facilitate Tuberculosis Drug Discovery
product using the Promega CellTiter 96 Non-radioactive Cell
Proliferation Assay.
Once the Level 2 screens are completed, select compounds are
considered for further evaluation in one of the following, the
macrophage and/or the single drug resistant strain assays. In the
macrophage assay, compounds are tested for killing of Mtb strain
Erdman (ATCC 35801) inside mouse bone marrow macrophages
[11] at 4-fold concentrations equivalent to 0.25, 1, 4 and 16 times
the MIC. EC90 and EC99 are determined as the lowest concentration
effecting a 90% and 99% reduction, respectively, in colony forming
units at seven days compared to drug-free controls. Compounds
with EC90 >16 x MIC are considered inactive in the model. In the
single drug resistant (SDR) strain assay, MICs are determined in the
MABA against strains of drug-resistant M. tuberculosis (each strain
resistant to a single TB drug). Typically, all compounds progressing
to this stage of screening are tested against Mtb strains resistant to
INH (ATCC 35822), rifampin (ATCC 35838), and one other drug
resistant strain (the latter determined by the compound type) as well
as the drug-sensitive strains H37Rv and Erdman. Generally,
compounds are considered for further evaluation if their MICs for
SDR strains are less than 10 X MIC for susceptible strains. At this
stage a medical chemistry team at SRI reviews the data, considering
characteristics such as SAR, solubility, and log P values, to
determine if evaluation in in vivo testing models is warranted.
DEVELOPMENT OF NEW TESTING TECHNOLOGY
Although significant advancements were made in rational drug
design, drug development will continue to rely on the empirical
discovery of new lead compounds and therapeutic entities. This fact
is true, at least in part, because a complete understanding of drug-
receptor interactions and all of the mechanisms by which a
therapeutic goal can be achieved are not known. In fact, as new
drug targets are developed, it has become increasingly important to
be able to screen a large number of compounds quickly in order to
identify lead compounds to which the rational design elements of
medicinal chemistry can be applied.
The advent of high throughput screening (HTS) along with
combinatorial chemistry technology for the generation of either
structurally diverse or focused libraries of compounds has brought
about a significant change in the philosophy and approach to new
drug discovery. This approach utilizes the advantages of high
throughput screening and rational drug design to generate new
types of drugs in shorter periods of time. Robotics have been
incorporated into the TAACF activities by the addition of an HTS
contract led by Tom Fletcher (N01-AI-15449) with capabilities for
biochemical target screening using validated assays as well as
growth inhibition assays of Mtb in microtiter plates. Level 1 and
Level 2 testing are now conducted in 384-well format which
dramatically increases throughput. Over a period of 3 months more
than 200,000 individual compounds were tested in a high-
throughput assay for antitubercular activity.
In addition to the technological enhancements afforded via
HTS, a programmatic advantage is gained. Whereas previous arran-
gements called for two separate steps to arrive at dose dependent
results (primary screening at a single concentration, followed by a
separate compound aliquot and secondary screening for MIC
determination), the HTS protocols allow for dose response analysis
in the first screen, if warranted. This approach has led to much
shorter turnaround times for preliminary evaluations.
SELECTION OF COMPOUNDS FOR ADVANCED STUDIES
TAACF medicinal chemists select candidate compounds to
consider for advanced screening in animal models that exhibit
sufficient selective activity in antitubercular and cytotoxicity
screening. In some cases compounds are promoted directly to in
vivo studies, while in other cases, governed by such factors as
chemical class, advanced in vitro testing is prerequisite. For
Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2 95
example, candidate compounds that exhibit selectivity and also
share structural similarities with known antitubercular agents are
subjected to dose dependent screening against drug resistant strains
of Mtb. The goal is to identify compounds that fail to inhibit SDR
strain growth and eliminate them from consideration, while
identifying and promoting for further evaluation compounds that
successfully inhibit the growth of SDR strains. Typically, all
compounds progressing to this stage of screening are tested against
Mtb strains resistant to INH (ATCC 35822), rifampin (ATCC
35838), and one other drug resistant strain (the latter determined by
the compound type) as well as the drug-sensitive strains H37Rv and
Erdman.
Colorado State University (CSU) TAACF facilities are located
in Fort Collins, CO and are led by Drs. Ian Orme and Anne
Lenaerts. CSU performs in vivo testing as for compounds it receives
from SRI. The purpose of this program is to provide a resource
whereby new experimental compounds can be tested for their
capacity to inhibit the growth of virulent Mtb in an in vivo aerosol
mouse infection model and evaluated for preliminary toxicity,
maximum tolerated dose, and bioavailability. After each test is
complete, results are returned to SRI for ultimate delivery to the
compound suppliers.
DETERMINATION OF ACTIVITY AGAINST BACTERIA
IN THE NONREPLICATING PERSISTENCE NRP STATE
As part of the TAACF group, scientist at the University of
Illinois, Chicago (Dr. Scott Franzblau, PI) developed a high throu-
ghput, luminescence-based low-oxygen-recovery assay (LORA) to
test antimicrobial agents against NRP-Mtb [12]. This system uses
cells containing a plasmid with an acetamidase promoter driving a
bacterial luciferase gene, allowing optical measurement of the final
data read out. Agents targeting the cell wall were not active against
NRP-Mtb as would be expected for non-growing cells [12].
Rifampicin, streptomycin, PA-824, MFX (alone among the tested
quinolones) and clofazimine were among the most active com-
pounds against NRP bacteria. This assay has proved instrumental in
identifying new leads series with potent activity on NRP-Mtb,
including some with potency in the sub
g/ml range.
DETERMINATION OF MAXIMUM TOLERATED DOSE
(MTD)
C57BL/6 female mice (6-8 weeks in age) are administered a
one-time dose (oral gavage) of the compound at concentrations of
100, 300, and 500 mg/kg. The compounds are dissolved in an app-
ropriate vehicle (EtOH, DMSO or methylcellulose), or administered
in a suspension if necessary. There are 3 animals per dose and the
mice are observed post-administration for 4 hours, again 6 hours
later, then twice daily for the duration of the study (1 week). If an
animal exhibits obvious signs of distress (hunched posture, ruffled
fur, etc.), it is euthanized. Mice are sacrificed day 7 post-adminis-
tration and the critical organs are observed for evidence of drug
toxicity. If abnormalities exist or there were other animals in the
same group which did not survive to day 7, the tissues from the
liver, heart, and kidneys are removed and placed into 10% formalin
solution. These fixed tissues are sectioned and examined for
abnormalities resulting from drug toxicity. The MTD (mg/kg) is the
highest dose that results in no adverse effects.
DETERMINATION OF BIOAVAILABILITY (pK
BIOASSAY)
This bioassay estimates the drug levels in mouse serum at
specific time points post oral dosing, using growth inhibition of
Mtb H37Rv as an indicator for drug activity [13]. This assay is
conducted in 96-well microtiter plates. Each assay plate also has
control lanes of wells containing drugs of known concentrations,
either with or without 10% normal mouse serum. For each com-
pound tested, three C57BL/6 mice are given drug orally at a dose
96 Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2
lower than the MTD (usually 300 mg/kg or less). At specified time
points after dosing (usually 30 minutes and 2.5 hours) mice are
bled. Blood samples are collected aseptically, and centrifuged in
serum separator tubes to collect serum. Serum samples from mice
dosed with drugs are serially diluted using serum from naïve mice
as diluent and subsequently added to the 96-well assay plate.
Optical density at 600 nm is measured every 3 to 4 days, up to day
14. Results are confirmed by visual inspection at 10 days. Inhibition
of bacterial growth in the bioassay (<50% of the control without
drugs) indicates sufficiently high concentrations of bioactive
compound in the bloodstream and oral bioavailability of the
compound.
IN VIVO EFFICACY TESTING
Currently the TAACF uses two mouse models for efficacy
testing of compounds against Mtb; the Gamma Interferon Knockout
(GKO-IFN gamma knockout mice in a C57BL/6 background)
model [14] and a standard (C57BL/6 mouse) Mtb model [15]. The
GKO model allows a more rapid, initial assessment of drug efficacy
in vivo, while the standard C57BL/6 mouse model is used to
evaluate Minimal Effective Dose (MED), synergism with other
compounds in drug combinations, and ability of new compounds to
sterilize infected tissues.
GAMMA INTERFERON KNOCKOUT (GKO) MODEL
Gamma interferon knockout mice (unable to control an
infection of Mtb) from a C57BL/6 background are infected by
aerosol infection with the Erdman strain utilizing the Glas-Col
Inhalation Exposure System. Three mice are sacrificed day 1 post-
infection to determine bacterial uptake. On day 15 post-infection, 5
mice are sacrificed to determine bacterial load in the lungs and
spleens. Therapy, administered via oral gavage (or intraperitoneal
injection) begins day 15 post-infection and continues for 9 days
until day 24. On day 24 post-infection the mice are sacrificed and
bacterial loads are determined. An INH control group, administered
via oral gavage at 25 mg/kg/day is included in each study. Log10
protection values >0.50 generally indicate activity. INH has
demonstrated log10 reduction of the bacterial load in the lungs
around 2.75.
MURINE AEROSOLIZED TB MODEL: STANDARD
ANIMAL MODEL (SAM)
Wild-type C57BL/6 mice are exposed to an aerosol of Mtb
Erdman, which deposits 50 to 100 bacilli into the lungs of the
animal [15]. The course of this infection is followed in the lungs
and spleen for 8-12 weeks by plating homogenates of harvested
organs [n=5] on nutrient agar and determining bacterial numbers.
As the growing infection is slowly controlled and contained, a peak
number of about log 6.0 colony forming units can be observed in
the infected lungs. Test compounds are administered to groups of
mice starting on day 21 post-inoculation. Typically these are
administered by oral gavage. At this point in the screening program
the supplier is consulted regarding available information on com-
pound formulation and/or animal bioavailability and toxicity data.
An additional group is given INH as a positive control. Any effect
the compound may be having on bacterial numbers is then assessed
at intermittent time intervals of treatment (e.g. 2, 4, and 8 weeks of
drug treatment) and compared to untreated control values on these
days. The data is expressed as the log10 (and as log10 reduction in
colony forming units) provided by a given dose of the compound
against the growth of the organism in the untreated control group.
Compounds with log10 protection of > 0.60 log reduction in colony
forming units are generally considered active in the model.
Statistical tests are also applied to the raw data to determine levels
of significance.
Goldman et al.
TECHNOLOGY TRANSFER SUPPORT
As part of the NIAID’s TB Drug Development Program, RTI
International provides technology transfer support for promising
anti-TB compounds that have been successfully screened through
the TAACF program. RTI was instrumental in the development of
PA-824, which is now in clinical trials.
CHEMICAL DIVERSITY
Compounds Acquired from Suppliers
The compounds acquired from various suppliers encompass a
wide-ranging diversity in chemical space. A majority of the
compounds acquired and screened are pure synthetic compounds
possessing drug-like characteristics. Some of the major classes
represented in the evaluated compounds include: nucleosides,
purines and pyrimidines, folates, amino acids, peptides, sugars and
carbohydrates, steroids, lipids, terpenes, alkaloids and other natural
products, various monocyclic and fused-ring heterocycles and a few
aliphatics. Analogs of known antibacterial and antitubercular drugs
were also submitted and tested.
Prestwick Library
The original Prestwick library of compounds consisted of 880
off-patent high-purity compounds carefully selected for structural
diversity, broad spectrum of activity covering several therapeutic
areas (neuropsychiatry to cardiology, immunology, antiinflam-
matory, analgesia, etc.), known safety and bioavailability in
humans. Over 85% of its compounds are marketed drugs. This
library was screened for antitubercular activity and the data are
reported on the TAACF website (www.taacf.org). The newer
Prestwick library contains 1120 compounds, which will also be
tested via the TAACF.
ChemBridge Library
A ChemBridge library of compounds (www.chembridge.com) was
acquired for the TAACF program and consists of 100,997
compounds carefully selected for diversity. The ChemBridge
library was analyzed for diversity and drug-like properties [16] and
ranked among the best libraries available. The computed properties
of these compounds are within the Lipinski criteria for drug-like
molecules. This library consists of the following classes of
compounds: pyrroles, furans, thiophenes, indoles and their benzo
analogs, isoindolines, imidazoles, pyrazoles, triazoles, isoxazoles,
thiazoles, oxadiazoles, thiadiazoles, pyridines, quinolines, pyrida-
zines, pyrimidines, pyrazines, quinazolines, quinoxalines, pyrro-
lidines, piperazines and morpholines, Fig. (1).
Supplier Diversity
As seen in Fig. (2), the majority of compounds (~87,000)
submitted to the TAACF program are from the United States
(~38,000) followed by Asia (~26,000) and Europe (~17,000).
However, a distinction can be made between the types of suppliers
from different regions of the world. The United States suppliers are
mostly non-academic groups, while Asian and European suppliers
are mostly academic groups (with India leading the way in
compound submissions from Asia).
The TAACF is mostly utilized by academic suppliers (60%)
and small pharmaceutical companies (25%) who lack the means to
set up their own antituburcular screening facilities. Still, large
pharmaceutical companies (2%) have participated in the program
most often to screen libraries of compounds. Various research
institutes (10%) have also contributed. It is expected that such large
pharmaceutical company submissions will increase given the
TAACF implementation of high-throughput screening protocols for
the in vitro assays and acceptance of libraries of microtiter plate
samples.
Programs to Facilitate Tuberculosis Drug Discovery Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2 97

Fig. (1). Diversity of chemicals represented in the ChemBridge library of compounds.

Overall Statistics
As of January 31st, 2007, the TAACF had received 86,964
compounds from various academic and non-academic suppliers. Of
those compounds, 82,100 compounds have been screened in the
primary assays (see description of in vitro screening above). Of the
compounds screened, 8,040 compounds have shown 90% or greater
growth inhibition in the Level 1 assay.
All compounds showing greater than 90% growth inhibition are
considered active in the primary screen and are queued for the
Level 2 testing. The compounds are first screened in the secondary
MIC assay to confirm the activity. A total of 7,127 compounds have
been tested in the secondary MIC assay. A distribution of MIC
results for these compounds is shown in Fig. (3). Only 2.0 % of the
compounds tested in Level one gave MICs > 12.5 g/ml
demonstrating that the Level one screening was highly accurate,
given the single dose testing. Over 1000 compounds gave MICs of
< 1.0 g/ml.
All compounds with confirmed activity (MIC < 6.25 g/ml) are
also screened in the cytotoxicity assay. A total of 3,733 compounds
were tested for cytotoxicity. The selectivity indices (SI) of the
compounds are determined as SI = IC50 / MIC. Of the compounds
screened for cytotoxicity, 780 showed good selectivity (SI
10)
and were considered for in vivo testing.

SELECTION OF COMPOUNDS FOR IN VIVO TESTING

Various data are used in evaluating whether a compound war-
rants testing in vivo for efficacy. One factor used is the selectivity
Fig. (2). Diversity of Supplier by Geographic Distribution and Affiliation.
98 Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2
Fig. (3). Distribution of MICs on Mtb from Supplier Provided Compounds.
index, where a cut of SI
10) is usually, but not absolutely,
required for in vivo testing. Other tests are available such as the
single drug resistant strain and macrophage screens. For compounds
considered structurally novel, these screens can be performed to
determine whether a compound warrants testing in vivo. Medicinal
chemists review all in vivo candidates and make recommendations
to the Project Officer. Approved compounds are then tested in the
in vivo assays listed above. To date, over 150 compounds have
advanced to in vivo testing (79 for Bioavailability, 113 for MTD,
141 for GKO (30 showing some activity), and 51 for the SAM (11
showing some activity). Compounds of interest are highlighted in
later sections of this review.
PUBLICATIONS SUPPORTED BY TAACF DERIVED DATA
The TAACF encourages compound suppliers to pursue the
publication of their results in conjunction with or separately from
the NIAID and the screening contractors. As part of the publication
process, compound suppliers consult with the TAACF Project
Officer and medicinal chemists to ensure that all parties are
represented accurately. As of January 2007, these efforts have
resulted in over 200 publications and presentations featuring
research data derived from TAACF sponsored testing. These
publications are listed on the TAACF website at http://www.taacf.
org/publications.htm. This list is updated each month.
NOVEL COMPOUND SERIES SUPPORTED BY THE
TAACF
Quinoxaline-N-oxides
One particularly interesting class of antimycobacterials to
emerge from the TAACF program are the quinoxaline 1,4-di-N-
oxides prepared in the Monge laboratory [17-25]. Although several
quinoxaline-N-oxides were evaluated as general antibacterials in the
past [25-27] and, when considered as vitamin K analogs were even
earlier suggested for possible treatment of tuberculosis [28], the
systematic investigation of these agents as anitmycobacterials did
not occur until the present studies. Quinoxalines and their various
mono- and di-N-oxide derivatives as a class display a broad range
of biological activities [25,29] and Monge and collaborators had
Goldman et al.
been studying their effects in hypoxic tumor cells where aromatic
N-oxides are known to undergo bioreduction [30,25]. In this
context, it seemed natural to evaluate this class against myco-
bacteria, given the known activity of other classes of bioreductive
agents (e.g. metronidazole, PA824 and OPC67683).
Over 500 quinoxaline derivatives from the Monge laboratory
have been tested by the program, including simple substituted
quinoxalines, 2,3-annulated quinoxalines, and their corresponding
1,4-di-N-oxides. Their synthetic accessibility has facilitated a wide-
ranging structure-activity analysis, with diverse functionality
incorporated primarily at the 2-, 3-, 6-, and 7-positions. Many of
these compounds possess excellent antitubercular activity, and the
range of possible substituents affords an opportunity to tailor both
the pharmacokinetic and activity profiles. Typical compounds
represented by structure I have MICs against Mtb H37Rv of ~0.10
g/ml and good selectivity according to the VERO cell model [22].
Select analogs were active on a panel of single-drug-resistant
strains and active in the TAACF macrophage model. Most
importantly, several of these analogs are active against NRP-Mtb in
the LORA assay. A number of these agents are currently being
evaluated in in vivo models by the TAACF and two analogs have
shown efficacy in vivo in the GKO model.
O O
R1 N
OCH3 1a: R1=H, R2=CH2CH3
b: R1=Cl, R2=CH2CH3
N R2 c: R1=H, R2=CH2C6H5
d: R1=Cl, R2=CH2C6H5
O
Structure I
Nucleoside Analogs
A number of interesting nucleoside analogs, particularly
adenosine derivatives, also possess antitubercular activity. For
example, Sartorelli and co-workers described several 2-fluorinated-
3-deazaadenosine derivatives with modest to good (< 10 g/ml)
potency [31]. These agents require mycobacterial adenosine kinase
Programs to Facilitate Tuberculosis Drug Discovery
for their activity, and functioned as selective substrates for the
mycobacterial enzyme over the human homolog. One compound
was active in the GKO model of Mtb infection. 2-Methyladenosine
displays similarly potent activity versus both Mtb and M. smeg-
matis [32,33] and like the 3-deaza analogs, also requires activation
by adenosine kinase for their activity. Design and evaluation of
substituted adenosine derivatives as potential antimycobacterials
remains an active area of research.
Gundersen et al. published the first report on the synthesis and
SAR study of the antitubercular activity of some new 6-arylpurines,
6-arylpurinenucleosides, and 6-arylpurinenucleoside analogs [34].
The 6-substitutions included 2-thienyl, 2-furyl, and 2-phenyl, while
the non-nucleoside 9- substituents included H, methyl, 3-allyl,
benzyl, cyclohexylmethyl, 2-phenylethyl, and tetrahydropyranyl.
The most active of these compounds was a 9-benzyl-2-chloro-6-(2-
furyl)purine which showed an MIC of 0.78
g/ml, low toxicity, and
good activity against several SDR strains of Mtb. While these
efforts have not resulted in a new TB drug, the positive data have
encouraged the Gundersen group to expand their SAR study of
these newly discovered lead compounds.
2,3-Diphenylindolizines
In a follow-up publication, Gundersen reported the synthesis
and test result of 1-substituted 2,3-diphenylindolizines, some of
which were previously prepared and studied as potential
antioxidants [35]. In this study, a group of 16 1-substituted analogs
of 2,3-diphenyl-7-indolizinecarbonitrile were synthesized, in many
cases requiring the development of new synthetic routes. The
evaluation of 2,3-diphenyl-7-indolizinecarbonitrile and these 16
analogs as antitubercular agents showed that only moderate activity
resulted with alkyl or acyl substituents at the 1-position. However,
good activity resulted with hydroxyalkyl substituents at the1-
position, with the most active compound being 1-[1-hydroxy-(4-
unsubstituted or 4-Cl- or 4-OH-phenyl)- methyl]-2,3-diphenyl-7-
indolizinecarbonitriles, which inhibited Mtb growth >90% at 6.25

g/ml. The significance of this study is that these indolizines were
the first reported to have antimycobacterial activity, thus illustrating
how the TAACF approach to the discovery of new antitubercular
agents is successful in identifying new potential agents.
Quinolones
MFX is a broad spectrum antibacterial agent in the fluoro-
quinolone (FQ) class that targets bacterial DNA gyrase [36] and see
Mdluli and Ma elsewhere in this issue. The in vitro activity of MFX
against a variety of mycobacteria including M. avium [37], M.
leprae [38] Mtb [39], M. kansasii [40], M. abscessus [41], M.
ulcerans [42], M. thermoresistibile [43], M. fortuitum [44], M.
marinum [45], M. kansasii [46], and M. chelonae [47] was
reported. MFX was screened both in vitro and in vivo against Mtb
in comparative studies with other fluoroquinolones and generally
rates among the most active of the clinically used gyrase inhibitors
[6,7,48-61]. Using a highly purified sample prepared from Avelox
tablets, the TAACF compared MFX to several other FQs and
experimental drugs [55-57] demonstrating very potent activity in
vivo. Alternative and effective delivery approaches for targeting
MFX to Mtb-infected cells have been reported [58]. Several
regimens have been proposed that replace current first line drugs in
newer protocols for the treatment of refractory forms of tuber-
culosis, and MFX appears to be bactericidal in treating patients with
tuberculosis [7,51,6,53,54,59,60]. As the fluoroquinolones have
found broader applicability in the community treatment of
mycobacterial infections, FQ resistance has risen [61-63]. In spite
of these issues, retrospective studies of patients treated for MDR-
TB suggest the FQ containing regimens are quite effective in curing
intractable forms of the disease [64].
Another novel quinolone, KRQ-10018 (II) was identified by
the TB Alliance-KRICT program (ICAAC poster, 2006, San
Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2 99
2
Francisco ) with the support of the TAACF (see Mdluli and Ma
elsewhere in this issue). Although less active than MFX in an in
vivo efficacy mouse model, more potent analogs were recently
identified.
O O
F
N OH
N
N
Structure II
Other quinolones such as gatifloxacin, MFX, and sitafloxacin
were evaluated as inhibitors of Mtb as well as other mycobacterial
species. In an effort to improve the activity of quinolones against
Mtb, Sriram and coworkers explored the incorporation of lipophilic
moieties on the amine group at the 7-position of the quinolones
[65]. A series of such ligands possessing the semicarbazony-
lisatinylmethyl group on the piperazine nitrogen of ciprofloxacin
displayed activity against Mtb H37RV with MICs in the range of
1.21–3.09 nM. Those compounds with MIC of < 2.0 nM were
nontoxic to Vero cells at 100 nM. Among the active compounds
(III) displayed no acute toxicity at the highest dose of 1000 mg/kg
in maximum tolerated dose (MTD) study. This compound displayed
moderate activity in the GKO mouse model, producing a 0.76 log10
reduction in colony forming units in the spleen, but insignificant
reduction in colony forming units in the lung.
NH2 O O
F
O NH
N O OH
N N
N N
Structure III
Mefloquine
This drug fits broadly into the arylmethanol class of antima-
larial drugs and is widely used for prophylaxis of chloroquine-
resistant Plasmodium falciparum. The antibacterial activity of
mefloquine (IV) drug versus Gram-positive bacteria was reported
[66] although the mechanism of antibacterial action is not well
delineated [67,68]. Mefloquine is also active in vitro and in vivo as
a single agent and in combination with other drugs against
Mycobacterium avium, and combinations with other drugs has been
suggested as an approach for the treatment of macrolide-resistant
MAC [69,37,70,68]. It is also notable that alternatives to macrolide-
containing regimens may have advantages for the treatment of
mycobacterial diseases in HIV patients where macrolides are
known to interfere with HAART protocols due to induction of
2
46th Interscience Conference on Antimicrobial Agents and Chemotherapy. Abstract
F1372: Preclinical Testing of the Quinolone KRQ-10018 for Activity against
Mycobacterium tuberculosis in a Series of In vitro and In vivo models, A. J. Lenaerts,
V. Gruppo, K. S. Marietta, C. M. Johnson, I. M. Orme.
100 Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2
O N
N
F
N
F F F
F Cl
F
Structure IV
hepatic P-450 enzymes and reduction of the effectiveness of HIV
protease inhibitors [69]. The antitubercular activity of mefloquine
has been assessed through the TAACF program and by others [67].
The isomeric mixture used for the treatment of malaria shows
modest activity against H37Rv with significant toxicity to Vero
cells in culture. The isolated (+) and (-) forms of the drug show
varying activities ranging from around 7
M for the (+)- and (-)-
erythro forms to 13-21
M for the (-)- and (+)-threo isomers,
respectively, in the microplate Alamar Blue assay [67]. The (+)-
erythro form was the most active and least toxic of the isomers, and
these results mirror findings from antimalarial studies. Analogs of
this drug and their antitubercular activity have been described
[67,66]. We recently reported that mefloquine was active in vivo in
3
the mouse model of TB infection and are pursuing further studies.
Aminohydrazones
US patent 3878201 (April 15, 1975) described the antitu-
bercular activity of novel aminohydrazones synthesized by
American Cyanamid Corporation. This work was brought to the
attention of the TAACF by the inventor, Dr. Andy Tomcufufik, and
a decision was made to remake and evaluate this compounds series,
as the original work included only preliminary studies of in vitro
and in vivo efficacy. Structures V, VI and VII were synthesized and
evaluated. These compounds were moderately potent with MICs in
the range of 1.0
g/ml. They showed no cross-resistance with PA-
824 and were active on several MDR-TB strains tested. Although
the selectivity index (IC50 for VERO cells / MIC) was less than 10,
these compounds were tested in vivo, since the original report
indicated some degree of efficacy (prolongation of survival) in mice
treated with the drug incorporated into the diet. Although some
toxicity was observed at 100 mg/kg dosing, efficacy was observed
(Table 2). In this experiment a decrease in the log colony forming
units of 0.5 was statistically valid.
N N
N
N Br
Cl Cl
Structure V: TAACF 294825
3
2006 Conference on Antimicrobial Resistance, Hyatt Regency Bethesda, June 26-28,
2006. Abstract P3: New Agents for Drug Resistant Tuberculosis (TB), R. Goldman, B.
Laughon, S. Franzblau, J. Krahenbuhl, L. Adams, A. Lenaerts, I. Orme, J. Secrist, L.
Young, K. Plumley, C. Lambros.
Goldman et al.
N
N
N Cl
Structure VI: TAACF 294826
Structure VII: TAACF 294827
ANALYSIS OF THE PRESTWICK CHEMICAL LIBRARY:
(SEE WWW.TAACF.ORG)
Sulfonamides
Sulfonamide antibacterials, as a class, are known to be gene-
rally ineffective as single agents for the treatment of tuberculosis,
although they have been used as monotherapies for the treatment of
skin infections caused by rapidly growing mycobacteria such as M.
fortuitum [71]. In early studies some of the sulfanilamides
displayed promising activities in vitro but failed to display
significant activity in vivo in animal models [72]. The Prestwick
library contains twenty sulfonamides that include marketed drugs
and other analogs. Among the sulfonamides, the veterinary agent,
sulfaquinoxaline sodium displayed the highest activity producing
93% inhibition of growth of Mtb at a concentration of 6.25 μg/ml.
Other sulfa drugs that displayed activity include sulfathiazole
(91%), sulfamonomethoxine (86%), sulfamethizole (76%),
sulfamethoxazole (65%), and sulfadimethoxine (63%).
Beta-Lactam Antibiotics
The Prestwick library contains a number of
-lactam antibiotics
belonging to the penicillin and cephalosporin class of compounds.
Not surprisingly, none of the 27 compounds present in the library
displayed any significant inhibition (> 90%) of growth of Mtb at a
concentration of 6.25 μg/ml. An early study demonstrates that
inhibition of mycobacterial growth by penicillin G and related
penicillanse-nonresistant penicillins occurred only at concentrations
exceeding clinically useful levels. Even with a
-lactamase resistant
penicillin such as oxacillin, a significant bacteriostatic effect was
observed only at a concentration of 25
g/ml [73]. The resistance of
Mtb to
-lactams is generally attributed to the production of
-
lactamase [74,75,32,76]. Other contributing factors for the
ineffectiveness of
-lactams include exclusion of the drug from the
site of action by an impermeable cell envelope and the low binding
affinity of the drugs towards the target penicillin-binding proteins
[74,75,32,76]. However, compounds such as meropenem [77] and
imipenem [78] have MICs in the range of a few
g/ml and addition
of -lactamase inhibitors can reduce the MIC of several drugs, such
as amoxicillin and ampicillin, to a few
g/ml [78,77,79].
Programs to Facilitate Tuberculosis Drug Discovery
Table 2. Activity of Aminohydrazones
TAACF No. MIC (
g/ml) SI (Selectivity Index)
294825 1.56 0.69
294826 0.78 2.17
294827 1.56 0.95
ND: Not determined.
Quinolones
Quinolones are known to have a broad antibacterial activity
spectrum against Gram-positive and Gram–negative pathogens
(including mycobacteria) [80]. Their good-to-moderate oral
absorption and tissue penetration, favorable pharmacokinetics in
humans, and excellent safety profiles have resulted in their being
used extensively in the treatment of infections. The quinolones
inhibit the function of bacterial DNA gyrase and topoisomerase IV.
The five active quinolones in the Prestwick collection are examples
of the newer, improved quinolones, whose development began in
the late 70s and which have better activity against both Gram-
positive and Gram-negative bacteria and better bioavailability as a
result of having 6-F and 7-piperazinyl substituents. The Prestwick
quinolones all showed high percent inhibitions in the TAACF
assay.
Tetracyclines
Tetracycline antibiotics are highly effective against a number of
Gram-positive and Gram-negative bacteria (in general, better
efficacy against the Gram-positives), rickettsiae, and many
chlamydial species. They inhibit bacterial protein synthesis by
binding to the bacterial ribosome and appear to require at least two
processes to gain access to the ribosomes in Gram-negative bacteria
(passive diffusion through the channels formed by the porin
proteins in the outer cell membrane, and an energy-dependent
active transport system, which pumps all tetracyclines through the
inner cytoplasmic membrane and which may also require a
periplasmic protein carrier [81]).
The TAACF evaluation of the tetracyclines in the Prestwick
collection showed that this broad spectrum activity also includes
activity against mycobacteria, as 5 of the 7 analogs showed in vitro
inhibitions of >90%. However, the antitubercular promise of
tetracyclines may be limited since historically most are bacterio-
static and toxic effects (gastrointestinal, phototoxic, renal, and
effects on calcified tissues) are commonly encountered after oral
administration of the drugs.
Aminoglycosides
The soil actinomycete-derived aminoglycosides are drugs
containing amino sugars linked to an aminocyclitol ring by
glycosidic bonds. They are polycations and their polarity is partly
responsible for their pharmacokinetic properties, which include
inadequate absorption after oral administration and rapid excretion
by the kidney. They are used primarily to treat infections caused by
aerobic Gram-negative bacteria, and interfere with protein synthesis
via a bacteriocidal mechanism [81].
The evaluation of the Prestwick aminoglycosides confirmed
that aminoglycosides have good activity against TB. Of the 12
Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2 101
In vivo efficacy in the GKO mouse model (log CFU reduction in organ)
25 mg/kg 50 mg/kg 100 mg/kg
Lung Spleen Lung Spleen Lung Spleen
ND ND ND ND -0.20 -0.32
0.01 -0.30 ND ND 0.99 0.33
0.62 -0.01 1.43 0.67 ND ND
aminoglycosides evaluated, streptomycin, dihydrostreptomycin, and
amikacin all had activities of >98%, suggesting that the latter two
may merit further study. Other complications of aminoglycosides
are their potential to produce reversible and irreversible vestibular,
cochlear, and renal toxicity, and thus, these drawbacks must be
overcome in any future antitubercular aminoglycosides. Although
the use of aminoglycosides is becoming more and more limited due
to resistance, there may be some opportunities for improvement.
Mtb does contain and express aminoglycoside modifying enzyme
activity [82,83,84,85] suggesting that the basal level of some
aminoglycosides might be set by a baseline level of drug
modification. It is conceivable that blocking this modification could
increase the potency of aminoglycosides for Mtb. In addition, novel
analogs might be discovered that regain binding to resistant
ribosome, as was done with macrolides via the ketolides.
Phenothiazines
The Prestwick screening set includes a variety of tricyclic
antidepressants and related compounds including eleven moderately
to highly active samples. The biological activity of the tricyclic
antidepressants and related compounds can be traced back to Paul
Ehrlich and his observation of the effectiveness of certain tricyclic
dyes in staining biological tissues [86]. Since that time, a wide
variety of activities have been ascribed to this broad class of
compounds including analgesic, anticancer and antitumor, CNS-
depressant, antihistaminic, and antibacterial [87]. Activity versus
bacteria and, in particular, mycobacteria has been noted in the
literature for several decades [86]. Implicated antibacterial
mechanisms include inhibition of calmodulin-like proteins [88],
specific photosensitizing activity [89], formation of charge transfer
complexes with vital cellular proteins [90], inhibition of a
mycobacterial type-II NADH-menaquinone oxidoreductase [91]
(see Teh, Yano and Rubin elsewhere in this issue) , inhibition of
mycobacterial phospholipases [92], and inhibition of mycobacterial
fabD the malonyl coenzyme A:acyl carrier protein tranacylase
[93,94]. There are several notable issues relating to the continued
pursuit of these compounds that include the need for more
definitive mechanistic information, particularly as it relates to the
typical activities of these compounds in the patient relative to
selectivity (e.g. separation of CNS depressant activity from
antibacterial activity). Recently, novel analogs with activity on Mtb
and with reduced binding to dopamine and serotonin receptors were
identified with the support of the TAACF [95]. Several new
derivatives with promising activity against Mtb [96,97,98,6,7] were
also reported. Further, definitive studies are needed relative to the
photo-activation of these types of compounds versus specific
targeting; for example the use of appropriate controls run in the
dark to verify that the activity relates to a specific target versus
inhibition by photosensitization and activated oxygen effects.
102 Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2
Antifungal Azoles
Data from the Prestwick library reinforces literature reports
relating to the potent activity of azoles versus the mycobacteria and
Mtb [99-102]. Six known azoles (miconazole, econazole, sulcona-
zole, isoconazole, clotrimazole, and ketoconazole) in the library
showed significant activity versus Mtb H37Rv in the Alamar Blue
assay. Most importantly, oral econazole therapy was efficacious in
a mouse model of tuberculosis [100] and econazole was active
against latent/persistent tuberculosis [102] and MDR-TB strains
[101]. Although the precise mechanism of these drugs versus the
mycobacteria is not known, the genome of Mtb encodes an
unusually high number of P450 enzymes (5) including CYP51 that
is highly homologous to the fungal 14
-lanosterol demethylase, the
target of the azole class of antifungal antibiotics [103,104].
Structural and inhibition studies suggest that CYP121 may also be a
good target for the azole antifungals in Mtb [105,106,107]. It has
also been proposed that inhibition of bacterial growth may in part
be due to effects on the biosynthesis of longer chain isoprenes and
menaquinone [108].
N
N
Cl
O SQ-109, and the novel diarylquinoline R207910) was supported by
Cl
Cl
Structure VIII: Econazole
Incorporation of this class into an antimycobacterial regimen
has been suggested as an inexpensive off label application that
could potentially address treatment of drug-resistant strains of Mtb
[100]. It is notable that reports relating to the treatment of M. avium
suggest that certain azoles in combination with rifabutin should be
avoided due to unfavorable side effects [109]. Since this class of
drug is common in first line therapies for tuberculosis, such
combinations should be carefully considered. It is notable that
econazole has been studied for a variety of indications in numerous
models of infection as well as in humans, and topical treatment of
bacterial infections with this drug has been reported [110].
Econazole is orally bioavailable in animals and humans and was
effectively distributed to most tested organs. An oral dose of 250
mg in adult humans gave average peak serum levels of 2-3
g/ml
between 2-3 hours post dosing. Single oral doses of 25 mg/kg in 4
children (aged 2.5 to 9 years) and 1.0 gm in eleven adults gave
rapid absorption with wide inter-patient variation in serum levels
(0.86 to 13.0
g/ml one hour after the first dose). Repeated daily
doses of 1 to 2 g in adults and 100 mg/kg in children, in a few cases
for periods of several months, gave serum levels similar to those
seen after single doses. Oral dosing was also efficacious for tested
fungal infections.
Anticancer Agents
Of the approximately thirty compounds within the Prestwick
library that are classified as anticancer agents, none of them appear
to have potent antitubercular activity. Those that had significant
percent inhibitions were the most cytotoxic compounds (8-
azaguanine, etoposide, daunorubicin, and dacarbazine). At the con-
centration utilized (10
g/ml), the modest effects seen by the other
anticancer agents are likely due to their inherent cytotoxicity.
Goldman et al.
Steroids
The Prestwick library contains nineteen compounds classified
as steroids or closely related compounds. In general these com-
pounds were devoid of antitubercular activity. Two compounds,
fusidic acid and tomatidine, had modest percent inhibitions (72 and
73, respectively). Tomatidine, a steroidal alkaloid, has some biolo-
gical activity in various systems, including antifungal [111],
anticancer [112] and antiprotozoal [113] activities. Fusidic acid is
an approved antibacterial agent in Europe and elsewhere, and is
available in topical and oral formulations. Its modest antitu-bercular
activity has been known since its initial isolation as a natural
product.
Work is in progress to determine if additional compounds from
the Prestwick library could have activity against Mtb in vivo as was
found for econazole, or serve as leads for more potent and selective
agents active against Mtb.
SUMMARY
The TAACF was developed to stimulate research into the
discovery of new drugs for treating tuberculosis not only by
providing needed services to research laboratories, but also by
developing new technologies and a centralized database of the
activity of novel compounds active against Mtb both in vitro and in
vivo. The development of three new antitubercular drugs, all of
which are in clinical trials (the nitroimidazole PA-824, the diamine
resources from the TAACF and NIAID. R207910 was recently
evaluated by the TAACF (A. Lenaerts et al) in guinea pigs and
found very active in reducing colony forming units and pathology
[114]. In addition NIAID supported studies demonstrated the
efficacy of MFX in animal models alone and in combination with
other TB drugs (National Institutes of Health contract AI40007,
grant AI58993), and these studies [115,53,116,6] set the stage for
combination drug trials in humans.
The need for and success of the program is demonstrated by 1)
the large number of investigators who are using the TAACF, 2) the
number of compounds submitted for testing, and most importantly,
3) the number of new compound series discovered to have activity
in vitro and in vivo. Several compounds suppliers are continuing to
pursue novel lead series identified as active by the TAACF, and
several are being evaluated in advanced animal models.
The adoption of HTS for testing antitubercular activity was
recently implemented at SRI and over 200,000 commercially
available compounds were tested in a single dose screen. The
TAACF is now in the process of determining MICs for the active
compounds, and following medicinal chemistry analysis and other
preliminary testing, these data will be made available via publi-
cation and the TAACF website. We anticipate that this endeavor
will identify several hundred compounds that meet select criteria
for advanced studies. Laboratories interested in submitting com-
pounds to the TAACF for testing may contact the Project Officer
(rgoldman@niaid.nih.gov), or visit the TAACF website (www.
taacf.org) to discuss particiaption with SRI staff.
Although there are several new compounds in early stage
clinical trials for TB (see Laurenzi, Ginsberg and Spigelman
elsewhere in this issue) the final outcome of clinical development is
not yet known. Thus, early stage, preclinical research needs to
continue especially for compounds that will dramatically shorten
therapy.
ACKNOWLEDGEMENTS
The TAACF is funded by the National Institute of Allergy and
Infectious Diseases contracts N01-AI-95364 (Principal Investigator
[PI] Jack Secrist), and NO1-AI 15449 (PI Thomas Fletcher III) the
Southern Research Institute, N01-AI-95385 (PI Ian Orme)
Colorado State University, N01-AI-95384 (PI Doris Rouse) to RTI
Programs to Facilitate Tuberculosis Drug Discovery Infectious Disorders - Drug Targets 2007, Vol. 7, No. 2 103

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Received: April 12, 2007 Accepted: April 23, 2007