Structure-Based Design of MptpB Inhibitors That Reduce Multidrug-Resistant Mycobacterium tuberculosis Survival and Infection Burden in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.8b00832

Mycobacterium tuberculosis protein-tyrosine-phosphatase B (MptpB) is a secreted virulence factor that subverts antimicrobial activity in the host. We report here the structure-based design of selective MptpB inhibitors that reduce survival of multidrug-resistant tuberculosis strains in macrophages and enhance killing efficacy by first-line antibiotics. Monotherapy with an orally bioavailable MptpB inhibitor reduces infection burden in acute and chronic guinea pig models and improves the overall pathology. Our findings provide a new paradigm for tuberculosis treatment.

Full text of DOI:10.1021/acs.jmedchem.8b00832

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Article
Structure-based design of MptpB inhibitors that reduce multi-drug-
resistant Mycobacterium tuberculosis survival and infection burden in vivo
Clare Vickers, Ana Silva, Ajanta Chakraborty, Paulina Fernandez, Natalia Kurepina, Charis
Saville, Yandi Naranjo, Miquel Pons, Laura Schnettger, Max Gutierrez, Steven Park, Barry
N. Kreiswirth, David S. Perlin, Eric J. Thomas, Jennifer S. Cavet, and Lydia Tabernero
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00832 • Publication Date (Web): 28 Aug 2018
Downloaded from http://pubs.acs.org on August 29, 2018
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Structure-based design of MptpB inhibitors that reduce multi-drug-resistant
Mycobacterium tuberculosis survival and infection burden in vivo
1, §Clare F. Vickers,
2, §
2
2
Ana Silva, Ajanta Chakraborty, Paulina Fernandez,
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Natalia Kurepina, Charis Saville, Yandi Naranjo, Miquel Pons, Laura S.
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Schnettger, Maximiliano G. Gutierrez, Steven Park, Barry N. Kreiswith, David
S. Perlin, Eric J. Thomas, Jennifer S. Cavet, and 2, *Lydia Tabernero.
1
2
Affiliations:
1
The School of Chemistry, University of Manchester, Manchester M13 9PL, UK
2
School of Biological Sciences, Faculty of Biology Medicine and Health, University
of Manchester, Manchester Academic Health Science Centre, Manchester, M13
9PT, UK.
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Departament de Química Inorgànica i Orgànica, Universitat de Barcelona,
Baldiri Reixac, 10-12, 08028 Barcelona, Spain.
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Host-pathogen interactions in tuberculosis laboratory, The Francis Crick
Institute, 1 Midland Road NW1 1AT, London, UK
5
Public Health Research Institute, New Jersey Medical School, Rutgers
University, 225 Warren Street, Newark, NJ 07103.
§
*
these authors have contributed equally to the study.
corresponding author: Lydia Tabernero (Lydia.Tabernero@manchester.ac.uk)
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Abstract
Mycobacterium tuberculosis protein-tyrosine-phosphatase B (MptpB) is a
secreted virulence factor that subverts antimicrobial activity in the host. We
report here the structure-based design of selective MptpB inhibitors that reduce
survival of multidrug-resistant tuberculosis strains in macrophages and enhance
killing efficacy by first-line antibiotics. Monotherapy with an orally bioavailable
MptpB inhibitor reduces infection burden in acute and chronic guinea-pig
models and improves the overall pathology. Our findings provide a new
paradigm for tuberculosis treatment.
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Introduction
Tuberculosis (TB) remains a major health problem and leading cause of death
worldwide. Antibiotic resistance is a main obstacle in the cure and eradication of
TB, with over half a million new cases of drug-resistant TB per year. More
alarming is the increasing number of extensive or untreatable drug-resistant TB
cases (WHO TB Report 2017). Critical to the pathogenesis of Mycobacterium
tuberculosis, the causative agent of TB, is the secretion of virulence factors that
subvert the innate immune response and prevent bacterial control by host
macrophages 1, 2. In this context, anti-virulence drugs targeting the pathogen
survival mechanisms may represent an efficient complementary strategy to
antibiotics to increase efficacy and assist in clearing the infection. Anti-virulence
strategies are now emerging as promising therapies to counterbalance antibiotic
resistance in a number of microbial infections including TB3, 4, 5, 6,7, and yet this is
a largely unexploited area in the clinic.
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One such virulence factor is the MptpB phosphatase that is secreted into the
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cytoplasm of host macrophages . MptpB is critical for M. tuberculosis intra-
macrophage survival and for persistence of the infection in animal models9, 10
,
and acts by both attenuating the bactericidal immune responses and promoting
host cell survival. We report here that selective inhibition of MptpB impairs
survival of multi-drug resistant (MDR) TB in human macrophages and reduces
infection burden in acute and chronic guinea-pig models. Furthermore,
inhibition of MptpB enhances mycobacterial killing by the first-line antibiotics
rifampicin (RIF) and isoniazid (INH). Previously we have reported that MptpB
dephosphorylates in vitro the key signalling lipids phosphatidylinositol 3-
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phosphate (PI3P) and phosphatidylinositol 3,5-biphosphate (PI(3,5)P2) . These
lipids control critical steps of phagolysosomal biogenesis and bacterial
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clearance . We hypothesised that inhibition of MptpB activity may thus restore
the intrinsic host response compromised by this virulence factor, offering a novel
therapeutic mechanism against M. tuberculosis infections. Since MptpB is not
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essential for extracellular growth , its inhibition could prove a distinct
therapeutic advantage over antibiotics as it would potentially inflict less
selective pressure and reduce acquired drug resistance. An additional advantage
is that to block the secreted MptpB there is no need for drug delivery across the
complex and poorly permeable mycobacterial cell-wall. The lack of a human
orthologue also makes MptpB an attractive drug target for specific and selective
therapy.
Crucially, we, and others, have demonstrated that MptpB inhibitors impair
mycobacterial survival in macrophages13,14,15, supporting our hypothesis. Our
initial isoxazole-based MptpB inhibitors displayed modest potency and
13
selectivity . Other reported potent MptpB inhibitors showed little efficacy in
animal models15 indicating that optimisation of both target affinity and
pharmacokinetics are needed to develop compounds with in vivo efficacy.
We report here the rational structure-based development of our initial C1
isoxazole inhibitor 13 to generate a new series of MptpB inhibitors with improved
potency, selectivity and cell activity. Furthermore, an analogue from this series
showed an excellent pharmacokinetics profile, oral bioavailability and in vivo
efficacy in guinea pig models of tuberculosis infection.
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Results and Discussion
The structure of MptpB16 has an unusually large active site, with a primary
phosphate-binding pocket (P1) and two unique secondary pockets (P2 and P3)
not present in human phosphatases. In the crystallographic structure of MptpB
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with an oxalylamino-methylene-thiophene sulphonamide inhibitor , the
oxalylamino group binds to P1 whereas the sulphonamide partially occupies P2.
Molecular docking of our C1 inhibitor13 indicated that the isoxazole group
binded to P1 and P3, but P2 remained unoccupied (Fig. 1). Our strategy to
develop this initial hit used a structure-based rational approach aimed to retain
binding at P1 and P3 (isoxazole warhead) whilst exploiting binding at P2 to
increase potency and selectivity, see, for example, structures 2 and 5 (Scheme 1,
Figure 1).
Scheme 1. Development of the new series of isoxazole-based compounds. The
P1/P3 binding isoxazole warhead was used as the starting core. Subsequent
addition of a 4-phenyl linker and the dichloro-phenol fragment generated the
parent compound 5 providing higher potency and selectivity over human
phosphatase PTP1B.
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5 0.9 ꢀM
Figure 1. Rational structure-based design of MptpB inhibitors. a) Active site
of MptpB with compounds C1 (cyan) and 5 (orange) docked. The isoxazole
head in C1 and 5 occupy the P1 pocket (P-loop) and neighbouring P3 pocket,
whereas the additional dichloro-phenol group in 5 occupies P2. b) The poor
activity of the isoxazole head alone and that of the intermediates of the series
confirms that binding at P2 is essential to achieve higher potency.
13
Computational screening of commercial fragment libraries using a genetic based
algorithm18, 19 and the structure of MptpB identified >300 motifs interacting at
P2, providing suitable starting points for the design of a new series of 4,5-
diarylisoxazole-3-carboxylic acids (Scheme 2).
17
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Scheme 2 Isoxazoles identified for synthesis.
Synthesis of 4,5-diarylisoxazole-3-carboxylic acids
Isoxazoles 1 – 13 were identified for synthesis (Scheme 2). The synthesis of 4,5-
diarylisoxazole-3-carboxylic acids 1 – 4 is outlined in Scheme 3. Methyl 5-
phenylisoxazole-3-carboxylate 14 is well known 20, 21 and was synthesized from
acetophenone by a slight modification of literature procedures (see
experimental). Following bromination of the methyl carboxylate 14, Suzuki
couplings of the bromoisoxazole 15 22 with phenyl-, 3-methoxyphenyl- and 3-
hydroxyphenyl-boronic acids gave the 4,5-diarylisoxazole-3-carboxylates 16 23,
17 and 18. Saponification of these, and the isoxazolecarboxylate 14, gave the
corresponding carboxylic acids 1 – 4.
The triflate 19 prepared from the phenol 18 was coupled with the boronate 21,
that was available from 4-bromo-2,6-dichlorophenol 20, to give the methyl 4,5-
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diarylisoxazole-3-carboxylate 22. This was hydrolysed to give the isoxazole-3-
carboxylic acid 5 ready for screening, see Scheme 4.
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Scheme 3. Synthesis of the 4,5-diarylisoxazole-3-carboxylic acids 1 - 4 Reagents
and conditions: i, NaOH, MeOH, THF, rt, 1 h (1, 65%; 2, 60%; 3, 50%; 4, 55%); ii,
NBS, TFA, heat under reflux 48 h (64%); iii, ArB(OH)
2
, Na
2
CO , DMF,
3
o
bis(triphenylphosphine)palladium(II) dichloride or Pd(Ph
3
P)
4
, 90 C, 1 – 3 h (16,
80%; 17, 42%; 18, 49%).
Scheme 4. Preparation of the isoxazole-3-carboxylic acid 5 Reagents and
o
conditions: i, (CF
3
SO
2
)
2
O, py., DCM, 0 C to rt, 90 min (94%); ii,
bis(pinacolato)diboron,
dichloride, dioxane, 90 C, 18 h (59%); iii, 19, 21, DMF, Na
[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)
o
o
2
CO
3
, Pd(PPh
3
)
4
, 90 C,
3 h (45%); iv, NaOH, MeOH, rt, 1 h (81%).
The analogous isoxazole-3-carboxylic acids 6 – 12 were similarly obtained from
the triflate 19 and the requisite aryl boronic acid or boronate. These were
commercially available except that they required the preparation of the
isoxazole 12. This was prepared from the corresponding bromide (see
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experimental). Analogous chemistry was used to prepare the 5-methylisoxazole-
3
-carboxylic acid 13 from the known methyl 4-bromo-5-methylisoxazole 24, see
Scheme 5.
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Scheme 5. Preparation of the 5-methylisoxazole-3-carboxylic acid 13 Reagents
o
and conditions: i, NBS, TFA, 0 C, heat under reflux, 16 h; ii, ArB(OH)
2
, aq.
o
o
NaHCO
3
, DMF, Pd(Ph
3
P)
2
Cl
P)
2
, 90 C, 5 h (28%); iii, (CF
, DMF, 90 C, 3 h; v, NaOH, MeOH, 0 C, 1 h (65%).
3
SO
2
)
2
O, py., DCM, 0 C to rt,
o
o
6
h (66%); iv, 21, Pd(Ph
3
4
Activity of the 4,5-diarylisoxazole-3-carboxylic acids and biochemical
validation
The initial isoxazole fragment alone 1 showed very poor inhibition, but the
introduction of a phenyl ring at the 4-position reduced the IC50 from >500 ꢀM 1
to 17 ꢀM 2. Extending the aromatic ring with a di-chlorophenol fragment,
identified in the computational screen, afforded a further reduction to 0.9 ꢀM 5.
Improved binding for the new compounds was accompanied with increased
selectivity over the human phosphatase hPTP1B from 1.6-fold for compound C1
to 5.9-fold for compound 2 and 141-fold for compound 5 (Fig. 1b), supporting
the notion that binding at P2 is critical for selectivity.
Computational docking analysis of the new series of compounds showed a good
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correlation between the estimated free energy of binding and the experimental
activity (Supplementary Table 1). Molecular docking confirmed that additional
binding at P2 was responsible for the significant increase in potency of 5
compared to compounds 2 - 4. Subsequently, we explored variations of the di-
chlorophenol fragment substituents in the derivatives 6 - 12 (Table 1).
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The double and triple substituted aromatic groups showed similar IC50 to 5. The
introduction of the NO group at the meta-position of the phenyl ring 12
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increased potency by 50 % with respect to 5 resulting in an IC50 of 0.4 μM, and
afforded an excellent selectivity over human phosphatases (900-fold for PTP1B)
and the M. tuberculosis phosphatase MptpA (Table 1).
Our models suggested that the para-OH group of the phenyl ring (P2 head), could
form hydrogen bonds with the Oε1 of E129 (Fig. 2a) and that the meta-NO
2
group in 12 could form additional interactions with R136 or H94 thus explaining
its increased potency. Mutational analysis of the P2 pocket residues validated
this mode of binding. Mutations of E129 to alanine showed a 2 to 5-fold decrease
in inhibition (Fig. 2b), while mutation of H94 or R136 had a milder effect (~2-
fold decrease).
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Table 1. Inhibitory activity of the 4,5-diarylisoxazole-3-carboxylic acids
and selectivity over M. tuberculosis and human phosphatases. Scaffold of the
new series of isoxazole-based compounds is shown at the top. Substitutions of
the dichloro-phenol head (red square) were explored during development of
compound 5. Activities (IC50 values in ꢀM) towards M. tuberculosis MptpB,
MptpA, and human PTP1B and Vaccinia H1-related (VHR) phosphatases are
shown.
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a
b
P3
Compound
WT
H94A
E129A
R136A
1.0 ± 0.2
2.3 ± 0.5
P2
E129
H94
5
7
0.92 ± 0.03 1.3 ± 0.3 2.4 ± 0.1
2.3 ± 0.7 2.4 ± 0.2 7.0 ± 1.2
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R136
K164
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1.2 ± 0.3 2.9 ± 0.8 5.9 ± 0.8
1.1 ± 0.1 2.2 ± 0.3 5.7 ± 0.9
2.6 ± 0.3
2.1 ± 0.1
R166
C160
P1
12
0.40 ± 0.05 1.0 ± 0.1 1.9 ± 0.1 0.78 ± 0.03
c
**
****
*
***
****
****
****
****
****
Figure 2. Binding at the P2 pocket is important for the efficacy of the new
series of MptpB inhibitors. a) Mode of binding of the representative compound
5 into the active site of MptpB, as suggested from the molecular docking. b)
Mutation of key interacting residues at the P2 pocket resulted in a loss of 2-5 fold
the IC50 values (ꢀM). c) Cell activity of the new series results in substantial (81-
8
7%) reduction in the mycobacterial (BCG) burden of infected mouse
macrophages (J774) 72 hours post infection, compared to DMSO-treated
macrophages. Reduction in bacterial burden is already observed at 24 hours
post-infection (Suppl. Fig.2). Plots represent the average CFUs (+SEM) per well
(
from a 96-well plate, see methods for details) of at least three independent
experiments, with statistical significance relative to the control (DMSO treated)
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established using one-way ANOVA, Dunnett’s test (****p-value < 0.0001,
**p=0.012). Inset shows viability of treated macrophages.
The new MptpB inhibitors reduce mycobacterial burden in macrophage
infections
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Next we tested the efficacy of the new compounds in reducing mycobacterial
survival in macrophage infections. The new series of compounds showed dose-
dependent efficacy in reducing intracellular mycobacterial (BCG) burden in
mouse macrophages (J774) up to 87% after 72h of infection compared to control
(DMSO treated) (Fig. 2c). These findings are consistent with MtpB inhibition
assisting bacterial clearance in host macrophages, even in the absence of INF-γ
activation (see methods). The new compounds show increased efficacy at 80 µM
(
except for 5) compared to our initial C1 isoxazole inhibitor, thus correlating
with the higher potency of the new series. Toxicity of the new compounds is low
as shown in the cell survival assays (Fig 2c inset), with only compound 10
showing a substantial effect (>23% reduction) on cell viability at > 100 ꢀM
doses.
The new series of compounds displayed moderate permeability and lipophilicity
(logD), good solubility, they were stable in plasma with moderate clearance in
human liver microsomes, and high plasma protein binding (Suppl. Table 2). They
also showed poor in vivo pharmacological properties in guinea-pigs, with a
bioavailability below 13% (Table 2). We then replaced the phenyl ring at
position 5 of the isoxazole by a methyl group to reduce the bulk and number of
rings in the compound, generating compound 13. The potency of compound 13,
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at 3 ꢀM, was lower than the best of the parent series of inhibitors 5 and 12 (by 3-
7
fold), possibly due to reduced hydrophobic interactions at the P3 pocket.
However, compound 13 showed very good kinetic solubility (200 ꢀM), and a
good PAMPA value (78.1 nM/s) suggesting a potential good cell penetration,
despite showing a higher lipophilicity than the parent series (logD 4) (see
Supplementary material for details). Importantly, compound 13 displayed
improved pharmacological properties, it is orally bioavailable and has an
excellent phamacokinetic profile (Table 2), therefore it was selected for further
evaluation of its cell activity and efficacy in animal models of infection.
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Table 2. Pharmacokinetic parameters for MptpB inhibitors. Compounds 3, 8,
9
and 13 were tested in guinea-pigs to determine their PK profile. Cmax was
observed at 0.5 hr after IP (intraperitoneal) dosing and at 0.25 hr after PO (oral)
dosing.
Compound
13 (lead)
5
8
9
Guinea pig
Guinea pig
Guinea pig
Guinea pig
Route
IP
Oral
IP
Oral
5
IP
1.25
746
1.3
Oral
3.5
IP
Oral
5
Dose (mg/kg)
4
8
2.5
2.5
197
1.4
399
C
max (ng/mL)
31519 111099 3714
1.4 5.1 0.7
BLQ
BLQ
BLQ
<4%
BLQ
BLQ
BLQ
<13%
BLQ
BLQ
BLQ
<2.5%
t1/2 (hr)
AUCinf (ng.hr/mL) 71854 230407 4968
Bioavailability 156%*
1742
!
*High bioavailability may be due to prolonged absorption after the oral dose
limiting the rate of elimination; BLQ, below limit of quantification.
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MptpB inhibitors reduce survival of multidrug-resistant strains in
macrophages and enhance killing by first-line antibiotics
Investigation of compound 13 desmonstrated that it also exhibits dose-
dependent efficacy in reducing intracellular mycobacterial (BCG) burden in
mouse macrophages (J774) up to 84% (Fig. 3b), yet it does not affect
extracellular bacterial growth (Fig 3c), thus confirming that inhibition
exclusively targets intracellular mycobacteria, as expected. Critically, treatment
with compound 13 also reduces the intracellular mycobacterial burden in
human macrophages (THP1), up to 63% when using a drug-susceptible M.
tuberculosis strain (H37Rv), or up to 74% when using a MDR strain (Beijing-”W”)
(Fig. 3d). A similar effect was observed for the initial C1 compound
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(
Supplementary Figure 1) demonstrating that efficacy in reducing MDR-TB
survival is a general quality of MptpB inhibitors.
To test whether compound 13 is compatible with first-line TB drugs, isoniazid
(INH) and rifampicin (RIF), we determined doses of these antibiotics that caused
<
0
25% reduction in the bacterial burden of macrophages (0.1ꢀg/ml for INH and
.3 ꢀg/ml for RIF, Fig. 3e). We then used these doses of INH and RIF in
combination with a low dose, 5 µM, of compound 13. The combination resulted
in > 93% reduction in bacterial burden (Fig. 3e). Thus, treatment in combination
with compound 13 enhances killing by current anti-tubercular drugs. This is an
important finding since tuberculosis treatments rely on drug combination
therapies to clear the infection.
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Figure 3. Compound 13 reduces intracellular bacterial burden of H37Rv
and MDR-TB in macrophage infections. a) Structure of compound 13. b)
Effect of dose-dependent treatment with compound 13 on mycobacterial burden
(
BCG) in infected mouse macrophages (J774) at 72 hours post infection (****p <
0.0001, ***p < 0.0004, *p < 0.04 by one-way ANOVA, Dunnett’s test). Inset shows
macrophage viability upon treatment with 13 (**p=0.01). c) Extracellular
growth of BCG is not affected by treatment with compound 13 (80 ꢀM) respect
to DMSO control. d) Treatment with compound 13 (20 or 100 ꢀM) reduces
intracellular survival of an MDR strain (Beijing-W) and of the drug-susceptible M.
tuberculosis H37Rv strain in human THP1 macrophages (grey). Controls on the
effect on extracellular bacterial growth are shown (black), **p=0.0021; ***
p=0.0002, ****p<0.0001 by unpaired t test). e) Treatment with compound 13
increases the antibacterial activities of RIF and INH antibiotics in infected
macrophages (BCG in J774 mouse macrophages, ****p < 0.0001, **p=0.0017, by
one-way ANOVA, Dunnett’s test). d and e) data are the mean CFUs at 72 hours
post infection of at least three biological replicates (+SEM). Limit of detection is <
1
0 cfu.
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MptpB inhibition alters phagosomal phosphoinositde-3-phosphate
dynamics during infection
1
1
MtptB dephosphorylates PI3P and PI(3,5)P2 in vitro , two important anchors of
Rab proteins that drive phagosomal maturation and clearance of infection.
However, we do not know its effect on cellular PI dynamics. We tested if
compound 13 affected PI3P dynamics on M. tuberculosis-phagosomes. For that,
we monitored PI3P localisation by live-cell imaging in macrophages expressing a
fluorescent PI3P-binding module (FYVE2X-EGFP) after infection with
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fluorescently labelled M. tuberculosis H37Rv . PI3P associated with M.
tuberculosis-phagosomes immediately after infection, but PI3P signal decreased
2
4
rapidly after 2-4 minutes as previously reported . However, in the 13-treated
macrophages, the peak of PI3P was prolonged up to 12 minutes (Fig. 4). The data
indicate that MptpB inhibition extends the presence of PI3P and its association
with M. tuberculosis-phagosomes, suggesting a role for MptpB in host
1
1
phosphoinositide metabolism as hypothesised from its in vitro activity .
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cpd13 (80 ꢀM)
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10 12 14 16 18 20
Time (min)
Figure 4. MptpB inhibition alters phagosomal PI3P dynamics. Spatio-
temporal dynamics of PI3P (as visualised by expression of EGFP-FYVE) on M.
tuberculosis containing phagosomes (top figure). The plot shows the quantitative
analysis of the association of EGFP-FYVE to PI3P at the phagosomal membrane,
during the first 20 minutes of phagocytosis (data from 3 independent
experiments). Treatment with 13 extends the peak of PI3P up to 12 min.
compared to the rapid decay of PI3P peak in the untreated phagosomes (DMSO
control). DMSO, control for untreated macrophages (RAW264.7); cpd 13,
macrophages treated with compound 13.
Monotherapy with an orally bioavailable MptpB inhibitor reduces infection
burden in acute and chronic guinea-pig models
In vivo profiling of compound 13 showed high exposure (Cmax 112 ꢀg/ml, AUC
2
30 ꢀg.hr/ml), long half-life (t1/2 5 hours), good oral availability and relevant
tissue distribution in guinea-pigs, upon parental and oral dosing (Table 2 and
Supplementary Fig. 3), making it suitable for efficacy studies in animal models of
infection. Tolerability studies with 13 at 50, or 100 mg/kg (dosing once daily for
7
days) showed no adverse drug effects and weight increases of >5% was
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observed in all animals during the tolerability trial.
Compound 13 was then assessed for efficacy as monotherapy in the acute and
chronic guinea-pig models of TB infection. For the acute infection, animals were
infected with 96 CFU (avg. ± 27 SEM), and after 24hrs, orally dosed once daily
with compound 13 for 4 weeks. Treatment resulted in a 0.9 log reduction of
bacterial burden in the lungs relative to vehicle. For the chronic infection, guinea
pigs were infected with 63 CFU (avg. ± 18 SEM), and treatments were orally
administered daily for 4 weeks starting at 28 days post infection. Treatment with
compound 13 resulted in at least 1 log reduction in bacterial burden in lungs and
spleens (Fig. 5a and Supplementary Table 3).
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Pathological differences were observed between 13 and vehicle treated groups
in both lungs and spleens (Fig 5b-e) from both the acute and chronic infection
studies. Although the total number of tubercles were similar in both 13 and
vehicle treated animals, larger tubercles were consistently present in all vehicle
group. Overall, less damage to the spleens and lungs were observed in the 13
treated group relative to the vehicle only group.
Inspection of the lungs and spleens after 56 days of infection revealed visible
necrotic lesions ranging from ~1 to 4 mm, with more consolidation observed in
the vehicle group than in the compound 13 treated group, and with organs
presenting up to 19% increase in weight respect to the 13 treated group (Suppl.
Table 4). The histopathological analysis of the vehicle treated lungs and spleens
displayed large and diffuse confluent granulomas with necrotising cores and
inflammation throughout the parenchyma, pathology typically associated with a
chronic infection. The compound 13 treated lungs and spleens showed an
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improvement in the pathology with fewer smaller lesions and reduced
inflammation both in lungs and spleens (Fig. 5e).
These results provide proof-of-concept that MptpB inhibitors, used as
monotherapy, can be effective in controlling TB infection in animal models,
despite not having direct bactericidal activity. Particularly significant for clinical
applications is the efficacy and improved pathology observed in the chronic
model because guinea-pigs develop a similar immunopathology and
inflammatory response to the infection as in humans, forming granulomas that
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contain persistent bacteria 25, 26
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d Vehicle lungs
Vehicle spleen
e Vehicle lungs
Vehicle spleen
Treated lungs Treated spleen
Treated lungs
Treated spleen
Figure 5. MptpB inhibition reduces bacterial burden in animal models and
improves pahtology. a) Efficacy of monotherapy treatment with 13 for 28 days
in the acute and chronic guinea-pig models of TB (female Hartley Duncan).
Treatment was with 13 (100 mg/kg)) or RIF (50mg/kg) orally administered
once daily; VEH, vehicle control. Bars represent the mean value (±SEM) of CFUs
from 4-5 animals. Statistical significance is indicated (* p < 0.05 by one-way
ANOVA, Dunnett’s test). b) and c) Gross pathological scoring of the TB infected
guinea-pig lungs (b) and spleens (c) based on Jain et al. The lungs and spleen
from individual animals were given a score from 1 to 4 based on the number and
size of tubercles, level of involvement, inflammation and necrosis. Bars
represent the mean value (±SEM) of CFUs from 4-5 animals. Statistical
significance is indicated (* p < 0.05 by one-way ANOVA, Dunnett’s test). d)
Representative images of tissues at 56 days post infection (chronic model)
showing reduction of the number granuloma in the compound 13 treated lungs
and spleen. e) Representative histopathological images from lungs and spleens
at 56 days post infection from (chronic model). Lungs and Spleens fixed in
neutral buffer formalin were sectioned (5 micron) and stained with hematoxylin
and eosin (H&E) and imaged at 10x magnification. Vehicle treated lungs and
spleen show an increased presence of granulomatous infiltration (black arrows)
and pathologic damage relative to compound 13 treated lungs and spleen. Bars
represent 2 mm.
2
7
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Conclusions
In summary, our structure-guided drug development approach, exploiting
unique features in the MptpB structure, has delivered an orally bioavailable
compound with excellent therapeutic properties. We have demonstrated that
MptpB inhibitors are selective, effective against MDR-TB and that they increase
the intracellular killing efficacy of first line antibiotics RIF and INH, indicating
their suitability for combination therapies. Notably, reduction of intracellular
survival occurs in the absence of macrophage pre-activation with IFNγ,
suggesting that this strategy may be particularly advantageous when impaired
macrophage activation fails to control the infection (i.e. immuno-compromised
patients).
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Importantly, our lead compound showed efficacy in reducing bacterial burden in
a clinically-relevant guinea-pig model of infection, as well as improvement in the
pathology of lungs and spleen. This is also consistent with the enhanced
pathology and reduced inflamation observed when the mptpB gene is mutated9.
This is the first proof of concept that MptpB inhibitors, used as monotherapy, can
1
5
significantly reduce infection burden. A previous study showed a mild effect (<
.5 log reduction) when both MptpA and MptpB inhibitors where added to a
0
cocktail of three first-line antibiotics, isoniazid− rifampicin−pyrazinamide (HRZ),
effectively a 5-drug combination. However, there was no effect on bacterial
burden when only MptpA or MptpB inhibitors were added to the HRZ cocktail.
No data were presented on monotherapy treatments with either MptpA or
MptpB inhibitors, thus no direct comparison can be made with our results.
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Renewed interest in developing anti-virulence agents for TB treatment
(
reviewed in3, 4, 5, 6,7) makes this study timely. Furthermore, MptpB inhibitors
could be developed into broad-spectrum anti-virulence drugs, as MptpB
orthologues are present in more than 50 human pathogens including C. difficile,
1
1
1
1
1
1
1
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E. faecalis, K. pneumoniae, Yesinia spp and L. monocytogenes 28,29
.
Anti-virulence drugs hold great promise for the management of TB infections.
The current challenges in eradicating TB include: prevention with the existing
BCG vaccine has limited efficacy; standard treatments with antibiotics are long,
complex and chronic drug exposure over a prolonged period is linked to
development of drug resistance; and host-directed therapies are difficult to
control because they are patient-dependent. Anti-virulence agents offer
advantages to overcome these challenges because their action is independent of
the host fitness and they have the potential to limit drug resistance. Our findings
suggest that MptpB inhibition offers a new paradigm for TB therapy with the
potential to treat MDR-TB, improve antibiotic efficacy and overall pathology.
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Experimental Methods
Synthetic Chemistry
General experimental details
0
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All reactions were carried out under an atmosphere of dry nitrogen unless
otherwise stated. Low-resolution mass spectra were recorded on a Micromass
Trio 200 spectrometer using electron impact (EI) ionisation or electrospray in
+
-
positive (ES ) or negative modes (ES ). High-resolution mass spectra were
recorded on a Kratos Concept IS spectrometer. Infrared spectra were recorded
on a Genesis FTIR as evaporated films on sodium chloride plates. Proton NMR
1
13
spectra ( H NMR) and carbon NMR spectra ( C NMR) were recorded on Bruker
(500 MHz), Varian Unity 500 (500 MHz), Varian INOVA 400 (400 MHz) or Varian
INOVA Unity 300 (300 MHz) spectrometers. Residual non-deuterated solvent
was used as internal standard. Chemical shifts (δ
million (ppm) downfield from tetramethylsilane (TMS).
Flash column chromatography was carried out using silica gel 60H from
H
and δ ) are quoted in parts per
C
o
o
Merck. Light petroleum refers to the fraction that boils between 40 C and 60 C
and was distilled prior to use. Ether refers to diethyl ether that was used without
further purification. Tetrahydrofuran was dried over sodium/benzophenone and
was distilled under a nitrogen atmosphere. DCM was dried over calcium hydride
and distilled under an atmosphere of nitrogen. All other reagents and solvents
were used as purchased unless otherwise stated. The purity of compounds
submitted for screening was determined to be greater than 95% by HPLC (see
supplementary data for copies of the HPLC traces)
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Journal of Medicinal Chemistry
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5-Phenylisoxazole-3-carboxylic acid (1); standard procedure 30 31
Aqueous sodium hydroxide (2 M, 2.7 mL, 5.4 mmol) was added to methyl 5-
phenylisoxazole-3-carboxylate 14 (219 mg, 1.08 mmol) in MeOH:THF (1:1 v/v,
6
0 mL) at rt and the mixture was stirred for 1 hour. Water was added (60 mL)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and the mixture acidified to pH 1 using aqueous hydrogen chloride (2 M) then
concentrated under reduced pressure to give a slurry that was extracted with
EtOAc (3 × 60 mL). The organic extracts were dried (NaSO ) and concentrated
4
under reduced pressure to give an off-white solid that was recrystallised (light
petroleum:EtOAc, 100:1 v/v) to give the title compound 1 as a white solid (132
o
31
o
mg, 0.7 mmol, 65%); mp: 162.8 – 163.8 C (lit.
160-161 C); TLC (light
petroleum:EtOAc, 50:50 v/v, with 0.1% TFA): R
f
= 0.3; HPLC (silica, EtOAc with
1
0
1
.1 % TFA): retention time 3.27 min, 100 %; H NMR (500 MHz, DMSO-d
6
):
δ
13
4.13 (br. s, 1H), 7.96 (d, J 8.0 Hz, 2H), 7.59-7.54 (m, 3 H), 7.44 (s, 1H); C NMR
): 170.8, 160.9, 157.8, 130.9, 129.3, 126.2, 125.8, 100.9; IR
film): 3126, 3055, 2611, 2512, 1703,1474, 1445, 1261, 995, 914, 820, 760, 686
(125 MHz, DMSO-d
6
δ
(
-
1
cm .
4,5-Diphenylisoxazole-3-carboxylic acid (2) 23
Following the procedure outlined for the preparation of carboxylic acid 1, methyl
4
,5-diphenylisoxazole-3-carboxylate 16 (90 mg, 0.32 mmol) in MeOH:THF (1:1
v/v, 6 mL) and aqueous sodium hydroxide (2 M, 0.8 mL, 1.6 mmol), after
recrystallisation (light petroleum:EtOAc, 100:1 v/v) gave the title compound 2 as
o
a white solid (51 mg, 0.2 mmol, 60%); mp: 134.5-135.3 C; TLC (light
petroleum:EtOAc, 50:50 v/v with 0.1% TFA): R = 0.36; HPLC (silica, EtOAc with
f
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0.1 % TFA): retention time 3.18 min, 100 %; H NMR (500 MHz, DMSO-d
6
):
177.9,
61.0 151.8, 130.0, 129.1, 128.6, 128.4, 126.6, 116.2; IR (film): 3058, 1711, 1429,
δ
1
1
3.65 (br. s, 1H), 7.51 - 7.32 (m, 10H); 13C NMR (125 MHz, DMSO-d
): δ
6
-1
-
-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1225, 968, 770, 693 cm ; LRMS (m/z, ES ): 220 ([M 45] , 100%); HRMS (m/z):
+
[
M + H] , calcd. for C16
H
12NO3, 266.0812; found, 266.0817.
4
-(3-Methoxyphenyl)-5-phenylisoxazole-3-carboxylic acid (3)
Following the procedure outlined for the preparation of carboxylic acid 1, 4-(3-
methoxyphenyl)-5-phenylisoxazole-3-carboxylate 17 (100 mg, 0.32 mmol) in
MeOH:THF (1:1 v/v, 6 mL) and aqueous sodium hydroxide (2 M, 0.8 mL, 1.6
mmol), after recrystallisation (MeOH:H
2
O, 50:1 v/v) gave the title compound 3 as
o
a white solid (47 mg, 0.16 mmol, 50%); mp: 162.5-163.5 C; TLC (light
petroleum:EtOAc, 50:50 v/v with 0.1% TFA): R = 0.25; HPLC (silica, EtOAc with
f
1
0
.1 % TFA): retention time 3.20 min, 100 %; H NMR (500 MHz, DMSO-d
6
): δ
1
1
3.94 (br. s, 1H), 7.49-7.42 (m, 5H), 7.35 (t, J 8.0 Hz, 1H), 7.01 (dd, J 2.0, 8.0 Hz,
1
3
H), 6.95 (m, 1H), 6.91 (d, J 7.5 Hz, 1H), 3.73 (s, 3H); C NMR (125 MHz, DMSO-
d
1
1
6
): δ 166.0, 160.9, 159.2, 156.3, 130.6, 130.2, 129.7, 129.0, 126.7, 126.4, 122.2,
15.9, 115.7, 113.8, 55.1; IR (film): 3154, 1727, 1621, 1580, 1439, 1276, 1227,
-1
-
189, 1095, 1042, 1008, 966, 878, 826, 805, 768, 689 cm ; LRMS (m/z, ES ): 250
-
-
-
(
[M 45] , 100%); HRMS (m/z, ES ): [M

CO
2
H] calcd. for C16
H12NO ,
2
2
50.0873; found, 250.0861.
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Page 27 of 59
Journal of Medicinal Chemistry
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4-(3-Hydroxyphenyl)-5-phenylisoxazole-3-carboxylic acid (4)
Following the procedure outlined for the preparation of carboxylic acid 1, methyl
4
-(3-hydroxyphenyl)-5-phenylisoxazole-3-carboxylate 18 (150 mg, 0.52 mmol)
in MeOH:THF (1:1 v/v, 10 mL) and aqueous sodium hydroxide (2 M, 1.5 mL, 3
mmol), after recrystallisation (MeOH:H O, 50:1 v/v) gave the title compound 4 as
a white solid (80 mg, 0.29 mmol, 55%); TLC (light petroleum:EtOAc, 50:50 v/v
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
with 0.1% TFA): R = 0.5; HPLC (silica, EtOAc with 0.1 % TFA): retention time
f
1
3
7
.22 min, 100 %; H NMR (500 MHz, DMSO-d
6
): δ 14.04 (br. s, 1H), 9.56 (s, 1H),
.49-7.41 (m, 5H), 7.22 (t, J 8.0 Hz, 1H), 6.81 (d, J 7.5 Hz, 1H), 6.75-6.72 (m, 2H);
IR (film): 3468, 3243, 3064, 1722, 1629, 1583, 1474, 1444, 1331, 1228, 1176,
-1
+
+
971, 867, 795, 766, 687 cm ; LRMS (m/z, ES ): 282 ([M + 1] , 100%); HRMS
+
+
(
m/z, ES ): [M + H] calcd. for C16H12NO4, 282.0761; found, 282.0753.
4
-(3′,5′-Dichloro-4′-hydroxy-3-biphenyl)-5-phenylisoxazole-3-carboxylic
acid (5)
Following the procedure outlined for the preparation of carboxylic acid 1, methyl
-(3′,5′-dichloro-4′-hydroxy-3-biphenyl)-5-phenylisoxazole-3-carboxylate 22
900 mg, 2.04 mmol) in MeOH:THF (25 mL, 4:1 v/v) and aqueous sodium
4
(
hydroxide (2 M, 5.11 mL, 10.2 mmol), after recrystallisation (light
petroleum:EtOAc, 10:1 v/v) gave the title compound 5 as a white solid (700 mg,
o
1
.65 mmol, 81%); mp: 187.5-188.0 C; TLC (light petroleum:EtOAc, 50:50 v/v
with 0.1% TFA): R = 0.5; HPLC (silica, EtOAc with 0.1 % TFA): retention time
f
1
2
.79 min, 100 %; H NMR (500 MHz, DMSO-d
6
): δ 14.02 (br. s, 1H), 10.35 (s, 1H)
): δ 166.3, 160.9, 156.2,
and 7.75-7.32 (m, 11H); 13C NMR (125 MHz, DMSO-d
6
148.6, 137.5, 132.4, 130.6, 129.6, 129.2, 129.0, 128.1, 126.8, 126.6, 126.5, 126.3,
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
122.8, 116.0; IR (film): 3056, 1717, 1468, 1430, 1291, 1161, 1024, 868, 794, 768,
-1
-
-
-
-
6
6
4
91 cm ; LRMS (m/z, ES ): 426 ([M   1] , 6%), 424 ([M   1] , 8), 382 ([M – 45] ,
-
-
-
35Cl
,
5), 380 ([M – 45] , 100); HRMS (m/z, ES ): [M  H] calcd. for C22
H
12NO
4
2
24.0148; found, 424.0154.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
4-(4′-Hydroxy-3-biphenyl)-5-phenylisoxazole-3-carboxylic acid (6)
Following the procedure outlined for the preparation of the methyl 4-
biphenylisoxazole-3-carboxylate 22, the aryl triflate 19 (300 mg, 0.7 mmol) and
4
-hydroxyphenylboronic acid (107 mg, 0.77 mmol) in DMF (7 mL) together with
Pd(PPh (8 mg, 0.007 mmol), after repeated chromatography (Et O:light
3
)
4
2
petroleum, 20:80 - 40:60 v/v, then with DCM) gave methyl 4-(4′-hydroxy-3-
biphenyl)-5-phenylisoxazole-3-carboxylate as a white solid (159 mg, 0.427
o
mmol, 61%); mp: 62.7-63.5 C; TLC (Et
2
O:light petroleum, 40:60 v/v): R = 0.14;
f
1H NMR (500 MHz, CDCl
): δ 7.61-7.57 (m, 3H), 7.54 (s, 1H), 7.48 (t, J 7.5 Hz, 1H),
3
7.43 (d, J 9.0 Hz, 2H), 7.39 (d, J 7.5 Hz, 1H), 7.36-7.33 (m, 2H), 7.29 (d, J 7.5 Hz,
13
1
1
1
1
H) 6.89 (d, J 8.5 Hz, 2H), 5.88 (s, 1H), 3.90 (s, 3H); C NMR (125 MHz, CDCl
3
): δ
67.4, 160.6, 155.7, 155.0, 141.3, 132.9, 130.5, 129.2, 129.1, 128.9, 128.5, 128.4,
28.3, 127.1, 126.9, 126.8, 116.8, 115.8, 52.9; IR (film): 3374, 1741, 1612, 1519,
-1
-
445, 1335, 1221, 1175, 1103, 1018, 838, 770, 694 cm ; LRMS (m/z, ES ): 370
-
+
([M – 1] , 100%); HRMS (m/z, ES ): [M + H]+ calcd. for C23
H
18NO , 372.1231;
4
found, 372.1223.
Following the procedure outlined for the preparation of the carboxylic
acid 1, methyl 4-(4′-hydroxy-3-biphenyl)-5-phenylisoxazole-3-carboxylate (160
mg, 0.43 mmol) in MeOH (4 mL) and aqueous sodium hydroxide (2 M, 1.1 mL,
2
.15 mmol), after recrystallisation (light petroleum:EtOAc, 100:1 v/v), gave the
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Page 29 of 59
Journal of Medicinal Chemistry
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o
title compound 6 as a white solid (90 mg, 0.25 mmol, 59%); mp: 186.0-187.3 C;
TLC (light petroleum:EtOAc, 50:50 v/v with 0.1% TFA): R
f
= 0.49; HPLC (silica,
1
EtOAc with 0.1 % TFA): retention time 3.23 min, 100 %; H NMR (400 MHz,
DMSO-d
H), 6.83 (d, J 8.5 Hz, 2H); 13C NMR (100 MHz, DMSO-d
156.3, 140.4, 130.7, 130.2, 129.4, 129.2, 129.1, 128.0, 127.8, 127.6, 126.8, 126.5,
6
): δ 13.99 (br. s, 1H), 9.60 (s, 1H), 7.67-7.41 (m, 10H), 7.25 (d, J 7.5 Hz,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6
): δ 166.2, 161.1, 157.4,
1
1
26.0, 116.2, 115.8; IR (film): 3058, 1754, 1655, 1612, 1545, 1512, 1441, 1222,
-
1
177, 836, 803, 760 cm .
4
-(3′-Hydroxy-3-biphenyl)-5-phenylisoxazole-3-carboxylic acid (7)
Following the procedure outlined for the preparation of the methyl 4-
biphenylisoxazole-3-carboxylate 22, the aryl triflate 19 (300 mg, 0.7 mmol) and
3
-hydroxyphenylboronic acid (107 mg, 0.77 mmol) in DMF (7 mL) together with
Pd(PPh (8 mg, 0.007 mmol), after repeated chromatography (Et O:light
3
)
4
2
petroleum, 20:80 – 40:60 v/v, then with DCM) gave methyl 4-(3′-hydroxy-3-
biphenyl)-5-phenylisoxazole-3-carboxylate as a white solid (50 mg, 0.135 mmol,
o
1
1
9%); mp: 59.0-59.6 C; TLC (Et
NMR (300 MHz, CDCl ): δ 7.54 (dt, J 2.0, 8.0 Hz, 1H), 7.48-7.44 (m, 3H), 7.38 (t, J
.0 Hz, 1H), 7.29-7.14 (m, 5H), 7.05-6.85 (m, 2H), 6.73 (ddd, J 1.0, 3.5, 8.0 Hz, 1H),
2
O:light petroleum, 40:60 v/v): R
f
= 0.14; H
3
8
1
3
5.92 (s, 1H), 3.76 (s, 3H); C NMR (100 MHz, CDCl
3
): δ 167.4, 160.4, 156.1, 154.8,
41.9, 141.1, 130.5, 129.9, 129.2, 129.1, 129.0, 128.9, 128.8, 127.3, 127.0, 126.6,
19.3, 116.6, 114.6, 114.1, 52.8; IR (film): 3393, 1741, 1597, 1445, 1332, 1220,
1
1
7
-1
-
-
+
+
83, 694 cm ; LRMS (m/z, ES ): 370 ([M – 1] , 80%); HRMS (m/z, ES ): [M + H]
calcd. for C23
H
18NO
4
, 372.1231; found, 372.1238.
ACS Paragon Plus Environment