Mycobacterium tuberculosis gyrase inhibitors as a new class of antitubercular drugs

Article abstract of DOI:10.1128/AAC.03913-14

One way to speed up the TB drug discovery process is to search for antitubercular activity among compound series that already possess some of the key properties needed in anti-infective drug discovery, such as whole-cell activity and oral absorption. Here, we present MGIs, a new series of Mycobacterium tuberculosis gyrase inhibitors, which stem from the long-term efforts GSK has dedicated to the discovery and development of novel bacterial topoisomerase inhibitors (NBTIs). The compounds identified were found to be devoid of fluoroquinolone (FQ) cross-resistance and seem to operate through a mechanism similar to that of the previously described NBTI GSK antibacterial drug candidate. The remarkable in vitro and in vivo antitubercular profiles showed by the hits has prompted us to further advance the MGI project to full lead optimization.

Full text of DOI:10.1128/AAC.03913-14

AAC Accepted Manuscript Posted Online 12 January 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.03913-14
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Mycobacterium tuberculosis Gyrase Inhibitors (MGI) as a New Class of
Antitubercular Drugs
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Delia Blanco , Esther Perez-Herran , Mónica Cacho , Lluís Ballell , Julia Castro ,
Rubén González del Río1, José Luis Lavandera1, Modesto J. Remuiñán1, Cindy
Richards2, Joaquin Rullas1, María Jesús Vázquez-Muñiz3, Ermias Woldu4, María Cleofé
Zapatero-González3, Iñigo Angulo-Barturen1, Alfonso Mendoza1, David Barros1.
1Diseases of the Developing World, GSK, Severo Ochoa 2, 28760 Tres Cantos, Madrid,
Spain.
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2 Antiviral DPU, Infectious Disease Therapeutic Area Unit, GSK, RTP, United States.
3 Molecular Discovery Research, GSK, Santiago Grisolía 4, 28760 Tres Cantos, Madrid,
Spain.
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4 TPV, RD Platform Technology & Science, GSK, RTP, United States.
Correspondent footnote
Present address of José Luis Lavandera: CEU. Institute of Applied Molecular Medicine
(IMMA), School of Medicine, Universidad CEU San Pablo, Campus Monteprincipe,
Boadilla del Monte, 28668 Madrid, Spain.
Address
monica.i.cacho@gsk.com.
D.B. and E.P. contributed equally to the work.
correspondence
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esther.x.perez-herran@gsk.com,
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ABSTRACT
One way to speed up the TB drug discovery process is to search for antitubercular
activity amongst compound series that already possess some of the key properties
needed in anti-infective drug discovery such as whole cell activity and oral absorption.
Herein we present MGI’s, a new series of Mycobacterium tuberculosis Gyrase
Inhibitors which stems from the long term efforts GSK has dedicated to the discovery
and development of Novel Bacterial Topoisomerase Inhibitors (NBTI). The compounds
identified were found to be devoid of fluoroquinolones (FQ) cross-resistance and seem
to operate through a mechanism similar to the previously described NBTI GSK
antibacterial drug candidate. The remarkable in vitro and in vivo antitubercular profile
showed by the hits has prompted us to further progress the MGI project to full lead
optimization.
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INTRODUCTION
Tuberculosis (TB), one of the oldest known infections, is still nowadays the second
leading cause of mortality worldwide (1). The World Health Organization (WHO)
estimated that there were 8.6 million new TB cases in 2012 and 1.3 million TB deaths,
with a particularly high incidence amongst HIV co-infected individuals (2).
The current standard Directly Observed Treatment Short-Course (DOTS) (3) consists of
2 months of treatment with Isoniazid (INH), Rifampicin (Rf), Pyrazinamide (PZA) and
Ethambutol (EMB) followed by 4 additional months of INH and Rf (4). Nevertheless,
resistance to Isoniazid as well as to the combination of Isoniazid and Rifampicin is
common, a situation defining multidrug-resistance (MDR). Additionally, the emergence
of novel strains which are resistant to these two drugs in addition to one of the three
most commonly employed injectables has given rise to a novel category, the
extensively drug-resistant (XDR) (5) strain. One step further, total drug resistance
(TDR), is defined by resistance to all second-line drug classes. TDR cases have been
increasingly reported in the clinic (6,7).
Over the past decade, drug discovery and development efforts have increased, fuelled
by the upcoming threat of drug resistance in combination with the expansion of the HIV
pandemic. These realities highlight an urgent need for a more effective and tolerable
treatments for drug susceptible and drug resistant disease in addition to latent TB
infection.
Taking advantage of the broad expertise of GSK in antibacterial drug discovery, a large
subset of compounds representative of the wide chemical diversity generated in the
GSK Novel Bacterial Topoisomerase Inhibitor (NBTI) initiative (8,9,10,11), were
evaluated in vitro against M. tuberculosis. This exercise resulted in the identification of
novel M. tuberculosis DNA Gyrase Inhibitors (MGI’s), a new family of promising
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compounds with potential for the treatment of TB disease already reported in the
literature as antimycobacterial and antibacterial agents (12,13,14,15). Recently
AstraZeneca has reported anti-TB activity for NBTIs (16).
Herein, we introduce MGIs as new advanced leads against TB as evidenced by their
attractive in vivo and in vitro antitubercular profiles in addition to their lack of cross-
resistance with FQs.
MATERIALS AND METHODS
General aspects and ethics Statement. All the experiments were approved by the
Diseases of the Developing World, GSK Ethical Committee. All animal studies were
ethically reviewed and carried out in accordance with European Directive 2010/63/EU
and the GSK Policy on the Care, Welfare and Treatment of Animals. Specific Pathogen-
free 6–8-week-old female C57BL/6j mice (18–20 g) are obtained from Harlan (Harlan
Interfauna Iberica, Spain). The experiments were performed at AAALAC-accredited
GSK Laboratory Animal Science animal facilities in Tres Cantos (Madrid, Spain). The
mice were kept in air-conditioned facilities with fifteen air changes per hour. Room
temperature and relative humidity were 2263uC and 40–70%, respectively. The mice
were accommodated in groups of up to five individuals in TecniplastH type IV cages
with autoclaved dust free corncob bedding (Panlab, Barcelona, Spain). The mice were
maintained under a twelve hours light/dark period. Autoclaved tap water and irradiated
pelleted diet were provided ad libitum. The compounds used in these in vivo studies
were prepared as suspensions in 1% Methyl Cellulose. The antitubercular standards
used in the efficacy study were: Moxifloxacin (Sequoia Research Products Ltd)
prepared as solution in 20% Captisol(R)/water).
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Compound synthesis. Compounds 1 (17), 2 and 3 (12) were prepared following a
previously described synthetic pathway, see in the supplemental material.
In vitro assays
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Bacterial strains and culture. M. tuberculosis strains (H37Rv, Beijing 1237,
CDC1551, Erdman and 8 clinical strains), Mycobacterium canetti and Mycobacterium
bovis BCG were grown at 37ºC in Middlebrook 7H9 broth supplemented with 0.025%
Tween 80 and 10% ADC or on Middlebrook 7H10 plates supplemented with 10%
OADC. Escherichia coli DH5α was grown in LB broth. Ampicillin, Hygromycin,
Sucrose reagents were used for plasmid selection or recombination experiments.
DNA manipulation, plasmids and transformation. General molecular biology
procedures were used as described previously (18) or following the manufacturer
instructions. Escherichia coli DH5α and M. tuberculosis H37Rv competent cells were
prepared for electroporation as described previously (19).
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MIC Determination. Mtb H37Rv ATCC 27294 was used for all the studies and was
grown and tested as described earlier (20). The minimum inhibitory concentration
(MIC) was determined as described previously (20).
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Killing assays. Bacteria were grown at 37ºC in 7H9 Middlebrook-ADC-Tween80 to
mid-exponenetial-exponential phase and then diluted in 10ml fresh Middlebrook 7H9-
ADC-Tween80 to 5x105 CFU/ml. Incubation was continued after the addition of
compounds at 20X the MIC. At specified time points, aliquots of cultures were
withdrawn, serially diluted in 7H9 Middlebrook -ADC-Tween80 and plated on solid
culture medium (7H10-OADC). Plates were then incubated at 37ºC and CFUs were
counted after 3 to 4 weeks.
Generation of spontaneous resistant mutant rate. The MICs in solid medium
Middlebrook 7H10-OADC were determined in 24 well plates with serial dilutions of the
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compound of interest. 5 μl of bacterial culture containing 105 CFU/ml was added per
well. Plates were incubated at 37ºC for 20 days for M. tuberculosis H37Rv. MICs
values are the minimum concentration of the compound which inhibits 90% bacterial
growth.
In order to determine the generation of spontaneous resistant mutants rate to the
compounds, bacteria were grown at 37ºC in 7H9 broth fresh Middlebrook 7H9-ADC-
Tween80 to mid-exponential phase and then diluted in fresh Middlebrook 7H9-ADC-
Tween80 to 5x108 cfus/ml. Middlebrook 7H10-OADC plates with 20xMIC of each
compounds were inoculated with 108,107, 106 and 105 CFU/plate and the plates were
incubated at 37ºC during 3-4 weeks. The frequency of appearance of resistant mutants
was calculated and isolated colonies were re-streaked onto new plates filled with
Middlebrook 7H10-OADC agar containing the drugs as well as on plates without the
drugs as growth control in order to isolate the colonies.
Characterization of M. tuberculosis H37Rv MGIs resistant mutants. Single colonies
were used to amplify and sequence the quinolone resistance determining region
(QRDR). DNAs of the 21 resistant mutants were extracted from liquid cultures grown
in Middlebrook 7H9-ADC-Tween80. 1ml of culture was centrifuged, the pellet
resuspended in 500 µl of distilled water and incubated at 90ºC for 2 h, and then filtrated
by using 0.22µm filters. 5 µl of the supernatant was used as a source of genomic DNA
for amplification of the QRDR. PCRs were performed by using the PuRe Taq Ready-
To-Go PCR Beads kit (GE Healthcare Life Sciences). QRDR was amplified with the
primers described (21): gyrA1 (5'-CAGCTACATCGACTATGCGA-3’) and gyrA2 (5'-
GGGCTICGGTGTACCTCAT-3’)
for
gyrA
and
gyrB1
(5'-
CCACCGACATCGGTGGATT-3’) and gyrB2 (5'-CTGCCAClTGAGTTTlGTACA-3’)
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for gyrB. Amplification was performed for 40 cycles (94º1’-55º1’-72º1’) to generate a
fragment of 320 bp for gyrA, and a fragment of 427bp for gyrB.
PCR products were purified and sequenced using the dRhodamine Terminator Cycle
Sequencing Ready Reaction kit (Applied Biosystems) in an ABI PRISM® 7700
Sequence Detection System (Applied Biosystems). The sequences obtained were
analyzed using Lasergene software (DNASTAR, DNA and protein sequence analysis
software).
Reversion from mutant to wild type gyrA. The system pNIL/pGOAL (22) was used.
Briefly, the gyrA wild type from H37Rv was amplified with the primers gyrA19 (5’-
GATCACTCCTAACACTCGTACCCGGC-3’)
and
gyrA20
(5’-
GATCGTGGGAGACCACCATGGATCCC-3’) and cloning in the pGEMt-easy
(Promega). A 3 kb NotI/ScaI fragment from pGEMt-gyrA was cloned into the NotI site
of p1NIL. The PacI cassette from pGOAL19 (hyg, PAg85-lacZ Phsp60-sacB) was then
cloned into the single PacI site to generate the suicide delivery vector pEGSK3. Vector
DNA was used to electroporate M. tuberculosis H37Rv. hygR kanR transformants
(single cross-overs) were inoculated in Middlebrock 7H9-ADC–Tween80 and incubated
2 weeks at 37ºC. Serial dilutions were plated onto 2% sucrose plates. Plates were
incubated for 3–4 weeks. sucR colonies were then streaked onto plates with and without
hygromycin or with and without MGI cpd to identify the different phenotypes.
Inhibition of Mtb DNA Gyrase by compounds. Mtb DNA Gyrase activity was
measured by supercoiling of pBR322 DNA (Inspiralis) similar to previously described
methods (23). 0.3 U of Mtb DNA Gyrase holoenzyme (Inspiralis) was used per reaction
(25 μL) as this was the amount required to almost fully relax 500 ng of pBR322 DNA
over 60 min at 37 ºC. In a total reaction volume of 25 μL, 0.3 U of Mtb DNA Gyrase in
50 mM Tris (pH 7.9) containing 5 mM DTT and 30% v/v glycerol was added to the
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reaction buffer containing 50 mM HEPES (pH 7.9), 6 mM magnesium acetate, 4 mM
DTT, 1 mM ATP, 100 mM potassium glutamate, 2 mM spermidine, 0.05 mg/mL BSA
and 500 ng relaxed pBR322 DNA. For inhibition studies, compounds were used at the
appropriate concentrations by diluting down from 10 mM master stocks prepared in
Me2SO (the final assay concentration was 1% v/v Me2SO for each compound dilution).
For negative controls, reactions were set up in the absence of Mtb DNA Gyrase. Each
experiment was done at least twice with similar results.
Several fluoroquinolones were tested as pharmacology tools. The fitted IC50 values for
Gatifloxacin (0.36±0.1 µM), Ciprofloxacin (4.90±0.8 µM), Moxifloxacin (2.50±0.8
µM) and Levofloxacin (5.15±0.8 µM) were in general consensus with literature data
(24).
In vivo efficacy assessment. Specific pathogen-free, 8-10 week-old female C57BL/6
mice were purchased from Harlan Laboratories and were allowed to acclimate for one
week. The experimental design has been previously described (25). In brief, mice were
intratracheally infected with 105 CFU/mouse (M. tuberculosis H37Rv strain). Products
were administered for 8 consecutive days starting one day after infection. Lungs were
harvested 24 hours after the last administration. All lung lobes were aseptically
removed, homogenized and frozen. Homogenates were plated in 10% OADC-7H11
medium for 14 days at 37ºC. All animal studies were ethically reviewed and carried out
in accordance with European Directive 2010/63/EU and the GSK Policy on the Care,
Welfare and Treatment of Animals.
Molecular modeling and docking calculations. M. tuberculosis Gyrase A homology
model was built using the S. aureus X-ray structure (PDB ID: 2XCS) available from
Protein Data Bank as template. Molecular modeling and energy-minimization
experiments were performed using MOE (26). For ligands, 3D structures were
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constructed using the Builder module of MOE and optimized by energy minimization
using MMFF94X force field. All the compounds were subjected to conformational
searches for their lowest energy conformations as docking input. Molecular docking
experiments were carried out with GOLD program (v4.01) (27). For each compound
the best pose was selected based on the evaluation their best scores and similar
interactions within the active site.
RESULTS
Screen of NBTI chemical diversity. In order to determine the potential of NBTI´s as
novel anti-tuberculars, a large subset of 3000 compounds representative of the wide
chemical diversity generated in the project (more than 10000 compounds) was selected
for evaluation in vitro against M. tuberculosis H37Rv.
The Mtb MIC results obtained showed a high hit rate of active compounds (68% of
them with MIC values lower than 10 M). The most potent derivatives matched or
improved the MIC values for currently used TB drugs, including last generation FQs.
Overall, 29% of the compounds had MICs < 1M and 18% MICs < 0.1 M.
Similarly to NBTIs, the general structure of TB active compounds was divided into
three different parts according to the three topologically important areas in terms of
interaction with the target Gyrase (8). First, a Left Hand Side (LHS) is responsible for
important contacts with the Gyrase DNA substrate. Second, a Right Hand Side (RHS)
finds itself imbedded into the Gyrase enzyme, in addition to potentially providing some
bacterial target selectivity. Last, a central Linker Unit (CU) establishes key interactions
with the Gyrase (8) and offers the possibility to modulate important physico-chemical
properties (solubility, CHIlogD) that can impact hERG inhibition (28).
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These initial structure-activity observations, inherited from the predecessor antibacterial
program, helped schematize synthesis (details to be reported elsewhere) in addition to
rationalize how to balance anti-mycobacterial potency with oral exposure, safety and
synthetic complexity. These efforts resulted in the identification of the 7-substituted-
1,5-naphthyridin-2-one core as a privileged LHS, the N-ethyl-4-aminopiperidines as a
linker, and monocyclic aromatic rings with different substitution patterns as the best
RHS option, in contrast to NBTIs bearing bicycles as RHS (15). These three favorable
features were combined in the synthesis of analogues 2 and 3 (Fig. 1).
Antitubercular profile. Compound 1, as the best NBTI direct screening representative
and novel synthetic entities 2 and 3 were selected to be progressed to a panel of in vitro
and in vivo studies to validate their potential as antituberculars. The extracellular MICs
were found to be 0.5 M for compound 1, 0.08 M for compound 2 and < 0.01 M for
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compound 3 (Moxifloxacin MIC = 0.15 M). In terms of intracellular antitubercular
activity against M. tuberculosis THP1 infected macrophages, these compounds have
shown a very good activity, MIC of < 0.02 M for compound 2 and 3.
MICs of the three key MGI compounds against a panel of M. tuberculosis clinical and
laboratory strains were then established (Tables 1 and 2) in order to have an early
indication of the potential of these compounds to address MDR strains. MICs were
found to be higher than for the reference M. tuberculosis H37Rv strain, while still
within a range acceptable for further MDR consideration.
As a next step in compound evaluation, the bactericidal potential of the new MGI
compounds was studied by determining the time-kill kinetic curves of Compounds 2
and 3 and compared with Linezolid as a bacteriostatic model drug while Isoniazid and a
GSK InhA direct inhibitor were used as a bactericidal control (Fig. 2). Results show that
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these news MGIs are bactericidal as defined by compound capable to reduce 99.9% of
the bacterial population in 7 days (29).
In terms of potential for spontaneous resistance mutant generation using compound 1 as
a model molecule, a key consideration in any target based program, the frequency of
spontaneous mutant generation was found to be 7.4x10-8 mutants/CFU, a number
clearly lower than Isoniazid and similar to that of Rifampicin. As a direct consequence
of these assays, twenty one independent spontaneous resistant mutant colonies to our
compounds were isolated and characterized. Sixteen of them had a single point mutation
in the quinolone resistance-determining regions (QRDR (30)). 13 were found in gyrA,
where 8 different mutations were identified (H87Q, P50T, E79G, D89N, R98H, S95G,
D89G and H52R), and 3 in gyrB, where two different mutations could be observed
(D495N and D495A). Only one of the mutations found in the QRDR of gyrA (D89N)
has been previously reported (31) as related to Fluoroquinolone resistance.
More in depth sequencing identified point mutations in gyrA but outside the QRDR in
the remaining 5 strains. The mutations were H368R, I348S and S178L (Fig. 3).
MICs in the spontaneous mutants were found to be between 10 and >200 times higher
than in the wild type with cross-resistance between the three described MGIs a common
finding not associated to FQs (Ciprofloxacin, Ofloxacin and Moxifloxacin). The only
exception was found in the D89 mutants, which show a slight but noticeable cross-
resistance (2-8-fold higher MIC for Ciprofloxacin and Moxifloxacin, compared to the
20-300-fold increase in MIC for our compounds) (Table 3).
As a further check on potential for cross-resistance in the clinic, five resistant mutants to
FQs were also isolated and characterized. In these mutants, the most commonly
encountered FQR mutations described in clinical isolates in gyrA (S91P, A90V and
D94G) (30, 31, 32, 33, 34, 35) were found. Of the laboratory mutants selected against
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FQs, only S91P mutants showed a slight cross-resistance with some of the lead MGIs,
while D94G and A90V mutants gave rise to a hypersensitive phenotype to most of the
MGIs tested.
In order to univocally correlate gene mutations with resistance, a system using a suicide
plasmid vector for mutant reversal (E79G, H87Q, S95G, H368R and I348S) was
performed. We used the system described by Parish and Stoker (21). Briefly, a plasmid
with the wild type M. tuberculosis H37Rv gyrA was transformed into the mutants and a
first homologous recombination took place. HygR recombinants were selected. A
second homologous recombination occurred and double recombinants are HygS-
SucroseR. Afterwards, gyrA was amplified and sequenced and MGI resistance
determined. As a example, In the case of E79G mutant the 15% of the HygS-SucroseR
transformants were sensible to our compounds and all of them have the wild type
phenotype (Table 4, Fig. S1). The same process was repeat with the other mutants
(H87Q, S95G, H368R and I348S) and there was a perfect correlation between loss of
mutation and reversion to sensitivity (data not shown).
Additionally, mode of action of a presentative set of NBTI and MGI compounds was
evalauted in the Mtb DNA Gyrase supercoiling assay. Determination of concentration-
dependent inhibitory effect of Compounds 1, 2 and 3 yielded the following IC50 values
2.80 ±0.65 µM, 10.98±3.41 µM and 5.4±1.2 nM respectivelly. Compound 2 exhibited a
worse correlation between Mtb DNA Gyrase IC50 and H37Rv MIC. Further studies are
necessary to address this behavior. Most of the compounds with MIC against M.
tuberculosis also inhibited the supercoiling activity of Mtb DNA Gyrase, thus
confirming their mode of inhibition (Fig. 4). Given the potential for toxicity due to
cross-inhibition with human Gyrase, a representation of this set was tested in the human
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topoisomerase II alpha (36). Results showed > 30-fold more potency in Mtb DNA
Gyrase than against the human enzyme.
Once the potential of the new MGI leads for MDR treatment had been clarified,
compounds 1, 2 and 3 were further assessed in an acute infection mouse model of
tuberculosis (25), measuring the fold reduction in colony-forming units (CFUs) in the
lung (Fig. 5) vs. untreated controls. A preliminary profiling of selected compounds
showed how the compounds were endowed with desirable pharmacokinetic and
toxicological profiles (Table 5). The fold reductions in CFUs were compared to that
achieved with gold standards drugs such as Isoniazid (25 mg/kg, po, uid) and
Moxifloxacin (100 mg/kg, po, uid). Both MGIs 2 (50 mg/kg, po, bid) and 3 (50 mg/kg,
sc, bid) showed a significant cfu reduction in the lungs of infected mice (> 2 logs
inhibition), showing potencies approaching the reference compounds. However, 1 (100
mg/kg, po, bid) a compound in theory representing a good balance between in vitro
anti-TB potency and pharmacokinetic profile (Table 5) failed to show activity at the
maximum dose tolerated. This lack of activity could due to worse intracellular
antitubercular profile exhibited by 1 compared with MGIs 2 and 3.
DISCUSSION
As a consequence of the long term efforts GSK has dedicated to the Novel Bacterial
Topoisomerase inhibitor (NBTI) field, new antibacterials with novel modes of action no
cross resistance with FQ, oral drug-likeness and good safety profile have been identified
(8,9,10,11). Taking this exercise as a starting point for TB drug discovery, a
representative set of this collection (3000 compounds) was screened against M.
tuberculosis resulting in the selection of compound leads 1-3 for further antitubercular
profiling. Compound 1 was identified as the best balanced NBTI both in terms of in
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vitro anti-TB potency and pharmacokinetic profile, while 2 and 3 were designed as
novel entities incorporating key lessons learned from the already known NBTI chemical
diversity.
The compounds tested were bactericidal in vitro (10 x MIC) and showed a low rate of
generation of spontaneous resistant mutants (similar to Rifampicin). As no evidence
was available to date supporting the fact that MGI compounds killed M. tuberculosis
through inhibition of the Mtb DNA Gyrase, and no assays were available for the TB
enzyme at that time, as an alternative strategy we sought to isolate laboratory resistant
mutants to these compounds both to determine if there were single point mutations in
the Mtb DNA Gyrase resistant mutants and to test for potential cross-resistance between
MGIs, NBTIs and FQs. All 21 MGI resistant strains isolated were found to harbour
mutations in either GyrA or GyrB. Selected gyrA mutant reversion to gyrA wild type
resulted in resistant phenotype reversal to sensitive, a situation affecting the 5 mutants
tested. These results strongly suggest that MGIs bind to DNA Gyrase proximal to the
FQ binding site, and that inhibition of this enzyme is the lethal event leading to the
bactericidal action of MGIs.
Interestingly, mutants outside the QRDR of gyrA have also been characterised. These
mutations could be responsible of changes in the conformation of the protein due to
steric impediments or interactions.
Recently, Mtb DNA Gyrase supercoiling assay was set up in house. Biochemical assay
demostred this class to exert its action via inhibition of Mtb DNA Gyrase.
During the preparation of this manuscript, it was described a close class of N-linked
aminopiperidinyl based Gyrase inhibitors by AstraZeneca (16). Mtb DNA Gyrase
supercoiling and the cleavage complex assays confirmed their mode of action.
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Following the identification of 1, 2 and 3 as potent anti-mycobacterials (Table 5), the
compounds were evaluated for their capacity to treat TB infection in a murine animal
model of infection. The in vivo antitubercular profile of compounds 2 and 3 showed a
significant reduction of the CFUs in the lungs of acutely infected mice in addition to
exhibiting a level of efficacy comparable to gold standards (Isoniazid and
Moxifloxacin) (Fig. 5).
The reported efforts resulted in the establishment of a TB specific lead optimization
effort. Current work is focused on the design of novel TB-tailored molecules that can
combine the numerous different factors governing successful drug design, i.e. in vitro
and in vivo potency, oral exposure, absence of cardiovascular liabilities related to hERG
inhibition (10,11) and lack of clastogenic potential deriving from cross-activity with
host topoisomerases. Further progress will be reported in due course.
ACKNOWLEDGMENTS
The research leading to these results has received funding from the Global Alliance for
TB Drug Development and from the European Commission Seventh Framework
Programme ORCHID project No. 261378.
We thank T. Kaneko, K. Mdluli, Z. Ma and C. Cooper (TB Alliance) for their helpful
discussions and expertise in the field. We would like to thank the Pharmacology
Department for providing the data presented. We would also like to thank all the
scientists from the NBTI team (Antibacterial DPU, Infectious Disease Therapeutic Area
Unit, GlaxoSmithKline) for their collaboration and help.
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FIGURE LEGENDS
FIG 1 Structure of best balanced NBTI/MGI hits.
FIG 2 Time-kill curves of M. tuberculosis H37Rv in 7H9ADC medium at 20xMIC of
the Compounds 2 and 3. Linezolid was used as a bacteriostatic model drug while
Isoniazid and a GSK InhA direct inhibitor as a bactericidal control.
FIG 3 Mapping of Resistance Mutations in GyrA-GyrB model from M. tuberculosis.
Mtb Gyrase A homology model was built using the S. aureus X-ray structure (PDB ID:
2XCS).
FIG 4 Plot of concentration-dependent inhibitory effect of a representation of MGIs and
NBTIs against Mtb DNA Gyrase expressed as pIC50 vs. H37Rv MIC (M). Compounds
1, 2 and 3 are indicated as black squares.
FIG 5 Antitubercular activity of Isoniazid, Moxifloxacin, and Compounds 1, 2 and 3 in
an acute infection murine model. Each dot represents data from an individual mouse.
DL: limit of detection (* p>0.05 was considered significant. Data analyzed by one
factor ANOVA and Games-Howell post hoc, since Levene test was p<0.05).
22

1
TABLE 1 MICs (M) of Compounds 1, 2 and 3 against 8 clinical strains of M. tuberculosis.
Compound
H37Rv Clin1
Clin2
3.66
0.56
0.11
0.08
0.3
Clin3
3.66
0.26
0.04
0.31
0.5
Clin4
1.22
0.26
0.04
0.12
0.3
Clin5
1.22
0.22
0.04
0.16
0.5
Clin6
ND
0.22
0.02
0.08
0.3
Clin7
4.88
0.09
0.02
0.31
0.6
Clin8
0.30
0.56
0.09
0.23
0.5
1
2
3
0.29
0.04
≤0.01
2.44
0.13
0.02
0.31
0.3
Ciprofloxacin 0.08
Ofloxacin
0.6
Moxifloxacin 0.1
0.08
0.04
0.06
0.04
0.06
0.04
0.08
0.04

2
TABLE 2 MICs of key MGI compounds against wild type M. tuberculosis laboratory strains.
MIC (g/ml)
Compound\Strain
H37Rv Beijing 1237 Canettii CDC1551 Erdman BCG
2
3
0.04
≤0.01 0.03
0.2
0.6
0.15
0.15
0.04
0.16
0.5
0.15
0.04
0.12
0.3
0.15
0.04
0.12
0.5
0.12
≤0.01
0.04
0.16
≤0.02
Ciprofloxacin
Ofloxacin
Moxifloxacin
0.08
0.3
0.08
0.04
0.08
0.08
0.08
3
4
5
6
7
8
9
10

11
TABLE 3 MIC values of the mutants are represented as the ratios of the MICs obtained for each compound in relation to the wild type.
RATIO MICmut/MICH37Rv
MGIR
FQR
GyrA
Mutation\
Compound
H87Q P50T R98H S95G D89G H52R E79G H368R I348S S178L
S91P D94G A90V
1
2
3
31
17
NT
2
10.7 31
11
8
NT
1
83
83
128
32
NT
2
4
2
21
8
11
8
NT
1.5
1
11
8
NT
1
NT N T
N T
0.1
1
8.3
NT
1
25
NT
1
>266 67
NT
3
1
>2
9
0.1
1
13.5
NT
2
1
NT
0.8
0.8
≥1
1
Ofloxacin
Moxifloxacin
9
1
1
1
2
3
1
NT N T
>25 74
25
25
1
Ciprofloxacin >1.5 ≥1
INH
≥1
1
>1.5 >3.8 >1.5
1
>1.5 ≥1
0.8
1
1
1
1
NT
1
1
1