The 7-phenyl benzoxaborole series is active against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.tube.2017.11.003

We identified a series of novel 7-phenyl benzoxaborole compounds with activity against Mycobacterium tuberculosis. Compounds had a range of activity with inhibitory concentrations (IC₉₀) as low as 5.1 μM and no cytotoxicity against eukaryotic cells (IC₅₀ > 50 μM). Compounds were active against intracellular mycobacteria cultured in THP-1 macrophages. We isolated and characterized resistant mutants with mutations in NADH dehydrogenase (Ndh) or the regulatory protein Mce3R. Mutations suggest that Ndh may be the target of this series.

Full text of DOI:10.1016/j.tube.2017.11.003

Accepted Manuscript
The 7-phenyl benzoxaborole series is active against Mycobacterium tuberculosis
Aaron Korkegian, Theresa O'Malley, Yi Xia, Yasheen Zhou, David S. Carter, Bjorn
Sunde, Lindsay Flint, Dean Thompson, Thomas R. Ioerger, Jim Sacchettini, M.R.K.
Alley, Tanya Parish
PII:
S1472-9792(17)30318-9
10.1016/j.tube.2017.11.003
YTUBE 1642
DOI:
Reference:
To appear in: Tuberculosis
Received Date: 26 July 2017
Revised Date: 28 October 2017
Accepted Date: 5 November 2017
Please cite this article as: Korkegian A, O'Malley T, Xia Y, Zhou Y, Carter DS, Sunde B, Flint L,
Thompson D, Ioerger TR, Sacchettini J, Alley MRK, Parish T, The 7-phenyl benzoxaborole series is
active against Mycobacterium tuberculosis, Tuberculosis (2017), doi: 10.1016/j.tube.2017.11.003.
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ACCEPTED MANUSCRIPT
The 7-phenyl benzoxaborole series is active against Mycobacterium tuberculosis
Authors:
Aaron Korkegian1, Theresa O’Malley1 Yi Xia2, Yasheen Zhou2, David S. Carter2, Bjorn
,
Sunde1, Lindsay Flint1, Dean Thompson1, Thomas R. Ioerger3, Jim Sacchettini3, M.R.K.
Alley2, Tanya Parish1*
1 TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, USA
2 Anacor Pharmaceuticals, Palo Alto, CA, USA
3 Texas A&M University, College Station, TX, USA
*Corresponding author:
tanya.parish@idri.org
Tel +1 206 858 6074
Fax +1 206 361 3678

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Abstract
We identified a series of novel 7-phenyl benzoxaborole compounds with activity against
Mycobacterium tuberculosis. Compounds had a range of activity with inhibitory
concentrations (IC90) as low as 5.1 µM and no cytotoxicity against eukaryotic cells (IC50
>50 µM). Compounds were active against intracellular mycobacteria cultured in THP-1
macrophages. We isolated and characterized resistant mutants with mutations in NADH
dehydrogenase (Ndh) or the regulatory protein Mce3R. Mutations suggest that Ndh may
be the target of this series.

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Tuberculosis (TB) represents a serious threat to global health as one of the top
10 causes of death worldwide with increasing rates of new infections (1). Current
treatments suffer from long treatment time and a growing problem of multidrug
resistance. There is therefore a need for new molecules with anti-tubercular activity as
well as new targets for the development of treatments.
The combination of two privileged structures (2), the biphenyls and the
benzoxaboroles identified a novel 7-phenyl benzoxaborole series with activity against
Mycobacterium tuberculosis in vitro. Three compounds with a 7-phenyl benzoxaborole
structure were tested for activity against Mycobacterium tuberculosis (Figure 1 and
Supplementary Information). Compounds had activity in both liquid and solid medium
(Table 1). We determined inhibitory concentrations under aerobic conditions as
previously described (3). Compounds were solubilized in DMSO and tested as 10-point
two-fold serial dilutions. Bacteria were cultured in Middlebrook 7H9 medium
supplemented with 10% v/v OADC (oleic acid, albumin, dextrose, catalase) and 0.05%
w/v Tween-80. Assay plates were inoculated with M. tuberculosis H37Rv LP (ATCC
25618) (4) and incubated for 5 days at 37ºC. Growth was measured using OD590 and
plotted using the Levenberg-Marquart algorithm. The IC90 was defined as the
concentration at which 90% growth was inhibited. All three compounds were active
against replicating bacteria. AN11987 had good activity, with an IC90 of 5.1 µM (Table
1). AN6288 and AN6291 had less activity, with IC90 of 80 µM and 55 µM respectively
(about 10-fold less active). Compounds were also active on solid medium. We
determined the MIC99 (defined as the concentration required to prevent 99% growth) for
each compound on Middlebrook 7H10 containing 10% v/v OADC. Again, AN11987 was

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the most active (MIC99 = 5 µM) as compared to AN6288 and AN6291 (MIC99 = 12.5
µM), although the difference was less pronounced (Table 1).
We also measured compound activity against intracellular bacteria. We infected
the THP-1 human monocytic cell line (ATCC-TIB202). Briefly THP-1 cells were
maintained in RPMI-1640, 10% v/v FBS, 2 mM Corning glutagro, 1 mM sodium
pyruvate and differentiated using 80 nM phorbol 12-myristate 13-acetate. Cells were
infected at a multiplicity of infection of 1 with M. tuberculosis constitutively expressing
luciferase (5), washed to remove extra-cellular bacteria and seeded into 96-well plates
containing compounds. Infected cells were cultured for 72 h and bacterial viability
assessed by RLU. The cytotoxicity of compounds was determined against uninfected
THP-1 cells exposed to compounds for 72h; cell viability was measured using CellTiter-
Glo (Promega). For both assays, curves were fitted using the Levenberg-Marquardt
algorithm. The IC50 and IC90 were defined as the concentrations that reduced
cell/bacterial viability by 50% or 90% respectively. All three compounds showed good
intracellular activity with no cytotoxicity (IC50 > 50 µM) (Table 1). The activity trend seen
in axenic culture was preserved against intracellular bacteria, with AN11987 being the
most active, IC90 = 1.8 µM. AN6288 was the least active, with IC90 = 30 µM. AN6291
had intermediate activity (IC90 = 5.0 µM).
Although the target for the 3-aminomethyl benzoxaboroles in M. tuberculosis is
leucyl-tRNA synthetase (LeuRS) (6) the target and/or mechanism of resistance for
these 7-phenyl benzoxaboroles compounds is unknown. We isolated resistant mutants
on solid medium (7). M. tuberculosis was plated onto solid medium containing 5X or
10X the MIC99. Colonies were isolated and confirmed for resistance in solid or liquid

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medium. We isolated and confirmed resistant mutants for AN6291 (7 mutants), AN6288
(2 mutants) and AN11987 (3 mutants). We selected three mutants resistant to AN6291
and two mutants resistant to AN6288 for whole genome sequencing.
Whole genome sequencing and mutation identification was conducting as
described in Ioerger et al 2010 (4) using an Illumina HiSeq 2500 and either 72x72bp
paired-end reads or 54x54 paired-end reads. We identified a number of single
nucleotide polymorphisms (SNPs) and a large deletion. Resistant mutants raised
against AN6288 contained mutations within ndh, while resistant mutants raised against
AN6291 contained mutations affecting Mce3R including an amino acid substitution, a
stop codon and a large deletion (Table 2). Mutations were confirmed by PCR
amplification and sequencing from genomic DNA. We sequenced ndh and mce3R in the
remaining isolates. All the strains isolated against AN6291 had mutations in mceR3,
whereas the strains isolated against AN6288 and AN11987 had mutations in ndh (Table
2).
We tested selected strains for cross-resistance; MICs were determined on solid
medium using a modified version of the MIC99, using 96-well plates and a smaller
inoculum to conserve compound. Again, we saw the same trend of activity with
AN11987 being most active (MIC = 0.2 µM) (Table 3). Cross-resistance to all three
compounds was observed in strains containing either ndh or mce3R mutations.
Ndh is an essential oxidoreductase, which catalyzes electron transfer from NADH
to menoquinone as part of the electron transport chain (8) . Ndh is the target of several
small molecule series including the phenothiazines (9) and quinolinyl pyrimidines (10).
Mutations in Ndh found in clinical isolates have also been associated with resistance to

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isoniazid (R13C, V18A, T110A, A226E, R268H, G313R and N316K) (11-13). The
mutations we identified (P223T, F421S, V332M and A333V) have not previously been
reported. However, alignment of M. tuberculosis Ndh with Staphylococcus aureus (pdb
4XDB) and Caldalkalibacillus thermarum (pdb 4NWZ) structures revealed that V332 and
A333 correspond to residues involved in the quinone binding pocket (14, 15). V332
(T315 in C. thermarum and T318 in S. aureus) is involved in NADH binding while A333
is (A316 in C. thermarum and A319 in S. aureus) is part of the conserved AQxAxQ motif
common amongst NDH-2 analogs. Given that these Ndh mutations conferred resistance
to all three compounds, we propose that Ndh is likely the target of this compound
series, and that mutations lead to loss of binding. Further work to express, purify and
co-crystallise the protein would address this question.
Mce3R is a TetR transcriptional repressor that negatively regulates genes
involved in lipid metabolism and redox reactions (16). The Mce3R mutations were
inactivating mutations, including premature stop codons, a frameshift and a large
deletion covering mce3R (Table 2). This suggests Mce3R might control genes involved
in resistance, since deletion of Mce3R activity would lead to upregulation of regulon
members. Genes controlled by Mce3R include a putative epoxide hydrolase (Rv1938),
a monooxygenase (Rv1936), a methyltransferase (Rv1498c) and a succinate-
semialdehyde dehydrogenase (Rv0234c) as well as several synthetases and conserved
hypotheticals of unknown function. Since MceR3 is a regulatory protein, we propose
that a regulon member might be involved in compound degradation or metabolism. It is
interesting to note that the addition of 6-methyl group to AN6291 yielded a more potent

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compound, while the mutations all mapped to ndh not mce3R, which suggest that this
methyl group might interfere with this resistance mechanism.
In conclusion, we identified a new benzoxaborole series, the 7-phenyl
benzoxaboroles, that had activity against M. tuberculosis in vitro and may target Ndh
rather than LeuRS. The 7-phenyl benzoxaboroles are effective against M. tuberculosis
in axenic culture and in macrophage culture, with no cytotoxicity. We characterized two
routes to resistance, one of which is via gene regulation (Mce3R) and the other in Ndh.
It is possible that ndh represents the target of the 7-phenyl benzoxaborole compounds
while Mce3R has some effect on the target or on the compounds through activity of a
gene within its regulon. We propose that this is a series with potential for further
development and target validation work.
We thank James Ahn and Yulia Ovechkina for technical assistance.
Funding
This research was supported with funding from the Bill and Melinda Gates Foundation
and by NIAID of the National Institutes of Health under award number R01AI099188.
The content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health

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Figure 1: Synthetic pathway of 7-phenyl benzoxaborole compounds.
R = H, X = Br; R = Me, X = I;a) (CH2O)n, MgCl2, TEA , MeCN, reflux, overnight, 90%; b)
MOMCl, DIEA, DCM, 0°C, 1h, 90%; c) 2-cyanophenylbor onic acid, Pd(PPh3)2Cl2,
Na2CO3, 1,4-dioxane, H2O, 80°C, 12 h, 46-56%; d) 2N HCl (aq), THF, 50°C, 4 h, 64-
93%; e) Tf2O, pyridine, DMAP, DCM, rt, 2h, 45-70%; f) (PinB)2, KOAc, Pd(PPh3)2Cl2,
1,4-dioxane, N2, 80°C, 6h, 67-87%; g) NaBH 4, MeOH, THF, rt, 0.5h; 6N HCl (aq), rt,
0.5h, 56-66%; h) NaOH, H2O, 80°C, 8h, 32-64%.

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Liquid
IC90 (µM)
80 ± 30 (n=4)
55 ± 13 (n=3)
5.1 ± 1.5 (n=4)
Solid
MIC99 (µM)
12.5
Intracellular activity
Cytotoxicity
IC50 (µM)
>50
Compound
AN6288
IC50 (µM)
15
IC90 (µM)
30
AN6291
AN11987
12.5
5.0
3.9
5.0
>50
>50
0.69
1.8
Table 1. Biological activity of compounds. Compounds were tested for activity in
liquid or solid medium. MIC99 is the concentration required to inhibit growth by 99%. IC50
and IC90 are the concentrations required to reduce bacterial/cell viability in the
intracellular macrophage activity assay by 50% and 90% respectively. Intracellular
activity is against M. tuberculosis, cytotoxicity is against THP-1 cells.

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Compound
Strain
Gene(s)
Mutation
Amino Acid change
AN6288
AN6288
RM1*
RM2*
ndh
ndh
667C>A
P223T
F421S
1262T>C
AN6291
RM1*
Rv1948c-Rv1970
deletion
AN6291
AN6291
AN6291
AN6291
AN6291
AN6291
AN11987
AN11987
AN11987
RM2
RM3
RM4
RM5*
RM6*
RM7
RM1
RM2
RM3
mce3R
mce3R
mce3R
mce3R
mce3R
mce3R
ndh
334T>C
+149-150 deletion
334T>C
W112R
W112R
C188F
Y185*
563G>T
555C>A
+649-936 deletion
994G>A
V332M
V332M
A333V
ndh
994G>A
ndh
998C>T
Table 2. Mutations identified in resistant isolates. Strains were isolated on 5X or
10X MIC on solid medium and were confirmed as resistant by determining MICs.
AN6291-RM1 had a large deletion encompassing mce3R. Mutations were identified by
whole genome sequencing in five strains (indicated by an asterisk). Mutations in the
remaining strains were identified by PCR amplification and sequencing of the gene.

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MIC solid (µM)
Amino acid
Mutation
Strain
Gene
change
AN6288 AN6291 AN11987
H37Rv
12.5
50
12.5
50
0.2
25
AN6288-RM1
AN6288-RM2
ndh
ndh
667C>A
P223T
F421S
1262T>C
50
50
3.1
Rv1948c-Rv1970
deletion
AN6291-RM1
*
50
100
3.1
AN6291-RM5
AN6291-RM6
mce3R
mce3R
563G>T
555C>A
C188F
Y185*
50
50
100
100
3.1
3.1
Table 3. Cross-resistance profile of resistant strains. MICs were measured on solid
medium in 96-well format. MICs were defined as the minimum concentration required to
inhibit growth completely. *AN6291-RM1 had a large deletion encompassing mce3R.