Discovery and Optimization of Two Eis Inhibitor Families as Kanamycin Adjuvants against Drug-Resistant M. tuberculosis

Article abstract of DOI:10.1021/acsmedchemlett.6b00261

Drug-resistant tuberculosis (TB) is a global threat and innovative approaches such as using adjuvants of anti-TB therapeutics are required to combat it. High-throughput screening yielded two lead scaffolds of inhibitors of Mycobacterium tuberculosis (Mtb)

Full text of DOI:10.1021/acsmedchemlett.6b00261

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Note
Discovery and optimization of two Eis inhibitor families as
kanamycin adjuvants against drug-resistant M. tuberculosis
Atefeh Garzan, Melisa J. Willby, Keith D. Green, Oleg V
Tsodikov, James E. Posey, and Sylvie Garneau-Tsodikova
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00261 • Publication Date (Web): 15 Sep 2016
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ACS Medicinal Chemistry Letters
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Discovery and optimization of two Eis inhibitor families as kana-
mycin adjuvants against drug-resistant M. tuberculosis
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Atefeh Garzan,a Melisa J. Willby,b Keith D. Green,a Oleg V. Tsodikov,a James E. Posey,b,* and Sylvie
GarneauꢀTsodikovaa,*
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536ꢀ0596, USA. b
Mycobacteriology Laboratory Branch, Division of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention,
Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
KEYWORDS Aminoglycoside acetyltransferase; Bacterial resistance; Enzyme inactivation; Drug combination; Structure-
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activity-relationship analysis.
ABSTRACT: Drugꢀresistant tuberculosis (TB) is a global threat, and innovative approaches such as using adjuvants of antiꢀTB
therapeutics are required to combat it. Highꢀthroughput screening yielded two lead scaffolds of inhibitors of Mycobacterium tuber-
culosis (Mtb) acetyltransferase Eis, whose upregulation causes resistance to the antiꢀTB drug kanamycin (KAN). Chemical optimiꢀ
zation on these scaffolds resulted in several potent Eis inhibitors. One compound restored the activity of KAN in a KANꢀresistant
Mtb strain. Model structures of Eisꢀinhibitor complexes explain the structureꢀactivityꢀrelationship. In the future, these Eis inhibitors
may potentially be developed into KAN adjuvant therapies against KANꢀresistant Mtb.
Despite extensive efforts to discover new antitubercular
agents in recent years, tuberculosis (TB) remains the top bacꢀ
terial cause of mortality worldwide. The proportion of new
multidrugꢀresistant (MDR)ꢀTB cases has not changed in recent
years. MDR strains of Mycobacterium tuberculosis (Mtb) are
resistant to the firstꢀline antituberculars isoniazid and rifamꢀ
picin. Extensively drugꢀresistant (XDR) strains of Mtb are
additionally resistant to fluoroquinolones and at least one of
the secondꢀline injectable antiꢀTB drugs, capreomycin, kanaꢀ
mycin (KAN), or amikacin. XDRꢀTB has very poor therapeuꢀ
tic outcomes. Therefore, the discovery and development of
new therapeutics is needed.
KAN is currently used to treat MDRꢀ and XDRꢀMtb infecꢀ
tions. Resistance to KAN observed in oneꢀthird of KANꢀ
resistant infections is due to the upregulation of the enhanced
intracellular survival (Eis) protein in Mtb.1 We previously
established that Eis modifies aminoglycosides (AGs),2 capreꢀ
omycin,3 and other lysineꢀcontaining biological molecules3 by
a unique multiacetylating mechanism.4ꢀ6 We also reported
crystal structures of EisꢀCoA complexes and that of an
Eis_MtbꢀCoAꢀtobramycin complex.7ꢀ10
New Eis inhibitors that can be used in combination with
AGs such as KAN is a potential way to overcome the reꢀ
sistance due to Eis upregulation. We reported some structural
scaffolds with inhibitory activity against the purified Eis enꢀ
zyme in vitro.11 Here, we report two new scaffolds: the methyl
4Hꢀfuro[3,2ꢀb]pyrroleꢀ5ꢀcarboxylate (scaffold 1) and the 3ꢀ
(1,3ꢀdioxolano)ꢀ2ꢀindolinone (scaffold 2) identified by highꢀ
throughput screening HTS, the synthesis of 13 and 14 anaꢀ
logues of these scaffolds, respectively, and their biochemical
and biological testing.
First, we screened ~123,000 small molecules for inhibition
of KAN acetylation by Eis_Mtb. The HTS yielded two promꢀ
ising scaffolds 1 and 2 (Fig. S1A). Sixty compounds containꢀ
ing scaffold 1 (Fig. S2) were present in the HTS library. In
these sixty compounds, the pyrrole ring was decorated with
different groups including alkyl chains, aryl and oxazole rings,
as well as amides. Fiftyꢀnine of these scaffold 1 molecules did
not inhibit Eis_Mtb, however compound 1a (Fig. S1B and
Scheme 1, also labeled as 4kk in Fig. S2) displayed some inꢀ
hibition in the HTS. Because 1a contained an aryl ketone
group, we opted to synthesize 1a along with 12 additional
analogues (1bꢀ1m) comprised of different aryl groups
(Scheme 1). For scaffold 2, ten compounds with different
groups attached to the indolinone ring were in the HTS library
(Fig. S3). A hydrogen (8a), an alkyl (8b), an alkyl ketone (8c),
a carboxylic acid (9a), or an amide (9b) substituent resulted in
no Eis_Mtb inhibitory activity. Similarly to the scaffold 1 anaꢀ
logues, aryl ketones of scaffold 2 (8fꢀ8h) displayed Eis_Mtb
inhibition, with the exception of the trifluoromethyl substitutꢀ
ed aryl ketones (8d and 8e). Three compounds synthesized
from scaffold 2 (8fꢀ8h, Fig. S3) were found to be active. These
and 11 of their analogues containing different aryl substituents
for further study (Note: 8fꢀ8h are numbered 2gꢀ2i in scheme 1
and Fig. S1 to represent the scaffold 2 series).
Compound 1a and 12 analogues (1bꢀ1m) as well as 14 anaꢀ
logues for scaffold 2 (2aꢀ2n) with different R substituents
were synthesized for structureꢀactivityꢀrelationship (SAR)
analysis of Eis_Mtb inhibition in vitro and in cellulo (Scheme
1
1). All new compounds were characterized by H, 13C NMR
(Figs. S5ꢀS56), mass spectrometry, and were established to be
≥95% pure by HPLC prior to further testing.
We evaluated biochemical (inhibition (IC50) of purified
Eis_Mtb enzyme) and biological (effect on the KAN MIC
values for KANꢀsensitive Mtb H37Rv and KANꢀresistant Mtb
K204 cells) properties of these compounds, in parallel (Table
1; Fig. S57). The freshly synthesized 1a, which displayed
some inhibition of Eis_Mtb in the HTS campaign, was conꢀ
firmed to be a good Eis_Mtb inhibitor in vitro (IC50 = 3 ± 1
ꢀM). In the presence of 1a, KAN displayed an MIC of 5ꢀ10
ꢀg/mL against Mtb K204. Having confirmed the weak inhibiꢀ
tory activity of 1a, we explored the effect of substitution on
the phenyl ring on the aryl ketone part of scaffold 1. Ortho
substitution, as in 1b with a oꢀfluoro substituent, resulted in
almost the same Eis_Mtb inhibitory activity (IC50 = 2.9 ± 0.9
ꢀM) as that for the parent 1a. KAN MIC against Mtb K204
was unaffected by 1b (MICKAN = 10 ꢀg/mL). To establish if
meta or para substitution would be more favorable than ortho
substitution, we generated compounds 1cꢀ1j. For both meta
and para substitutions, bulkier substituents led to weaker
Eis_Mtb inhibition (IC50 > 200 ꢀM for mꢀmethoxy (1f) and pꢀ
bromo (1i) compared to IC50 = 0.16 ± 0.07 and 0.3 ± 0.1 ꢀM
for mꢀfluoro (1c) and pꢀfluoro (1g), respectively). Most of
these derivatives (1cꢀ1i) did not improve KAN activity against
Mtb K204. The pꢀmethyl derivative 1j displayed almost the
same Eis_Mtb inhibitory activity (IC50 = 5.8 ± 1.8 ꢀM) and
MIC value against Mtb K204 as that of 1a. We generated 1k
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(naphthyl substituted) with the hope of strengthening any posꢀ
sible π−π interaction between the inhibitor and the AGꢀ
binding site of the Mtb Eis. This compound was found to be
completely inactive (IC50 > 200 ꢀM and MICKAN = 10 ꢀg/mL
against Mtb K204). Finally, replacing the phenyl ring with
alkyl chains (ethyl (1l) and tꢀbutyl (1m)) did not improve the
Eis inhibition or MICKAN for K204 Mtb.
sence of antibacterial activity of these compounds when used
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alone along with a general correlation between IC50 and MIC
values indicated that inhibition of Eis by these compounds is
the main mechanism of sensitization to KAN.
Table 1. IC50 values against purified Eis_Mtb and MIC values against
Mtb H37Rv and Mtb K204 with the compounds at the concentrations
specified.
IC50 (ꢀM)a
Cpd
Concentration H37Rv MICKAN K204 MICKAN
tested (ꢀM)b
(ꢀg/mL)c
≤1.25
(ꢀg/mL)d
≥10, 10
5, 10
-
ꢀ
3 ± 1
9
1a
1b
1c
1d
1e
1f
1g
1h
1i
100
100
15.5
25.1
52.6
100
33.9
64.5
100
100
100
100
100
32.6
100
29.7
1.5
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
≤ 1.25
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2.9 ± 0.9
0.16 ± 0.07
0.25 ± 0.08
0.5± 0.2
>200
0.3 ± 0.1
0.6 ± 0.3
>200
10, 10
10, 10
≥10, 10
≥10, 10
≥10, 10
10, 10
10, ≥10
≥10, ≥10
5, 10
10, 10
≥10, 10
≥10, 10
5, 5
1j
1k
1l
5.8 ± 1.8
>200
>200
1m
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
>200
0.33 ± 0.16
23 ± 10
0.30 ± 0.08
0.015 ± 0.005
0.54 ± 0.25
>200
0.09 ± 0.03
2.2 ± 0.7
>200
8.7 ± 2.2
>200
>200
>200
1.2 ± 0.4
10, 10
5, 2.5
≥10, 10
10, 5
Scheme 1. Preparation of potential Eis inhibitors scaffold 1 scaffold 2
core structures generated in this study.
For scaffold 2, compound 2gꢀ2i, which displayed Eis_Mtb
inhibition in the HTS, were freshly synthesized. The pꢀfluoro
54.2
100
8.9
≥10, 10
5, 5
substituted 2g displayed good Eis inhibitory activity (IC50
=
100
100
100
100
100
100
100
10, 5
≥10, 10
5, 10
10, 10
≥10, 10
≥10, 10
10, 5
0.09 ± 0.03 ꢀM) and when used in combination with KAN
resulted in MICKAN of 5 ꢀg/mL against K204 Mtb. The pꢀ
chloro substituted 2h was less active (IC50 = 2.2 ± 0.7 ꢀM)
than the pꢀfluoro substituted 2g and did not sensitize Mtb
K204 to KAN (MICKAN = 5ꢀ10 g/mL). The pꢀbromo substitutꢀ
ed 2i was found to be completely inactive (IC50 > 200 ꢀM and
MICKAN ≥10 ꢀg/mL against Mtb K204), while it displayed
limited Eis inhibition in the HTS, which could indicate that the
compound in the HTS library was not completely pure. We
also found that the pꢀmethyl derivative 2j displayed a 100ꢀfold
decrease in Eis inhibitory activity (IC50 = 8.7 ± 2.2 ꢀM) from
2g and in combination with KAN resulted in almost the same
KAN MIC (5ꢀ10 ꢀg/mL) against KANꢀresistant Mtb as 2g did.
The nonꢀsubstituted counterpart of parent 2g, derivative 2a,
displayed weaker Eis inhibitory activity (IC50 = 0.33 ± 0.16
ꢀM) and improved the activity of KAN against Mtb K204
(MICKAN = 5 ꢀg/mL). We also synthesized the mꢀfluoro, mꢀ
chloro, and mꢀbromo derivatives 2c, 2d, and 2e. The mꢀchloro
substituted 2d showed the same inhibitory activity as that of
2g, but did not sensitize Mtb K204 to KAN (MICKAN ≥10
ꢀg/mL). The mꢀfluoro and mꢀbromo substituted 2c and 2e
aIC50 against purified Eis_Mtb enzyme, bConcentrations of Eis inhibitor in
the MIC assays. At these concentrations, these compounds did not inhibit
the growth of Mtb H37Rv or that of Mtb K204 when tested in the absence
of KAN. Concentrations of Eis inhibitors were 100x their IC50 when IC50
<1 ꢀM, or 100 ꢀM for IC50 >1 ꢀM. cActivity of KAN against Mtb
H37Rv. dActivity of KAN against Mtb K204. For c and d, results are from
two experiments.
To investigate the selectivity of our inhibitors towards
Eis_Mtb, we tested two of our derivatives, one from each seꢀ
ries, 1c and 2c, against three other AAC enzymes with differꢀ
ent acetylation regiospecificities: AAC(2')ꢀIc from Mtb,5
AAC(3)ꢀIV from E. coli,12 and AAC(6')ꢀIe/APH(2")ꢀIa from
Staphylococcus aureus.13 Similarly to other known nonꢀEis
AACs, these three enzymes were previously shown to be
strictly regiospecific, but, like Eis, each enzyme was capable
of acetylating structurally distinct AGs. Neither 1c nor 2c inꢀ
hibited KAN acetylation by these three AACs at concentraꢀ
tions as high as 200 ꢀM, which indicated that our compounds
were highly selective against Eis_Mtb.
To explain the results of our SAR study, we used previously
published crystal structures of ternary Eis_MtbꢀCoAꢀ
compound A (13g in ref 14; PDB ID 5EC4) and B (11c in ref
14; PDB ID 5EBV) complexes to model our inhibitors 1a and
2g in a position similar to that of inhibitors A and B (Fig. S4).
Without the crystal structures, de novo computational modelꢀ
ing of and screening for Eis inhibitors, including pharmacoꢀ
phoreꢀbased computer modeling, are invalidated by significant
conformational changes in the Eis active site upon inhibitor
binding.13 The inhibitors occupy the site overlapping with the
AGꢀbinding site of Eis. The models show that the cores of
inhibitors 1a and 2g are surrounded by the side chains of hyꢀ
drophobic amino acid residues (Trp36, the aliphatic part of
resulted in a 3ꢀ and 5ꢀfold worse Eis inhibitory activity (IC50
=
0.30 ± 0.08 and 0.54 ± 0.25 ꢀM), respectively. When used
with the mꢀbromoꢀsubstituted 2e, KAN had an MIC of 5ꢀ10
ꢀg/mL against Mtb K204. However, when used with the mꢀ
fluoro substituted 2c, KAN displayed a better MIC value of
2.5ꢀ5 ꢀg/mL. As observed with scaffold 1, the presence of mꢀ
methoxyꢀphenyl, naphthyl, ethyl, and tꢀbutyl groups in scafꢀ
fold 2 resulted in molecules that were completely inactive
(IC50 > 200 ꢀM and MICKAN ≥10 ꢀg/mL against Mtb K204).
For scaffold 2, we also synthesized a mꢀnitro substituted comꢀ
pound (2n) to investigate the potential effect of a strong elecꢀ
tronꢀwithdrawing group on Eis inhibitory activity. Compound
2n was less active (IC50 = 1.2 ± 0.4 ꢀM) than 2g, but it sensiꢀ
tized Mtb K204 to KAN (MICKAN = 5ꢀ10 ꢀg/mL). The abꢀ
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Glu401, Ile28, Phe24, and Val400). The cores stack with the
Corresponding Author
* Eꢀmail: sylviegtsodikova@uky.edu; Phone: 859ꢀ218ꢀ1686;
FAX: 859ꢀ257ꢀ7585 or Eꢀmail: jposey@cdc.gov
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indole of Trp36, and in the orthogonal direction they are
sandwiched between Glu401, the Eis Cꢀterminus on one side
and Ile28 on the other. The acetophenone rings of both series 1
and 2 with different substituents stack with Phe84, explaining
why replacing these aromatic rings with alkyl chains resulted
in a loss of activity for 1l, 1m, 2l, and 2m. The acetophenone
rings are also surrounded by several hydrophobic amino acid
residues (Phe84, Trp36, Met65, Ala33). Therefore putting a
polar methoxy group in this hydrophobic environment would
likely destabilize Eis binding, explaining the IC50 values of
>200 ꢀM for 1f and 2f. The para position of the acetophenone
rings is flanked by Phe84 and Trp36, and it is ~5 Å away from
Trp13 and Met65, explaining why the bulkier bromo substituꢀ
ents of 1i and 2i resulted in lower Eis inhibitory activity,
whereas the small fluoro substituents of 1g and 2g improved
Eis inhibitory activity. The ortho position of the acetophenone
rings is flanked by Phe402, explaining why an ortho substituꢀ
ent, as in 1b and 2b, resulted in a loss of Eis inhibition. The
models shows that there is space for small substitution in the
meta position of the acetophenone rings (a ~5 Åꢀgap). Small
substituents such as the fluoro and chloro of 1c, 1d, 2c and 2d
fit well in the cavity, explaining why these compounds disꢀ
played good Eis inhibition. However, bulkier substituents such
as the bromo of 1e and 2e or the nitro of 2n are too big to be
accommodated at this site and would clash with Eis residues,
accounting for the poor Eis inhibition by these compounds.
We also determined that the calculated LogP values of all
compounds are in the desirable range (0.98ꢀ3.75; Table S1).
In sum, we have discovered two scaffolds with Eis inhibitoꢀ
ry activity. From 27 synthesized analogues of these scaffolds
with the variable acetophenone appendage, we identified poꢀ
tent inhibitors of Eis. Growth inhibition studies of our inhibiꢀ
tors in combination with KAN in KANꢀsusceptible Mtb
H37Rv (MICKAN ≤1.25 ꢀg/mL) and KANꢀresistant Mtb K204
(MICKAN ≥ 10 ꢀg/mL) showed that some of our inhibitors
were able to sensitize Mtb K204 to KAN. Smaller substituents,
like hydrogen and fluorine, yielded the best compounds. In
contrast, larger substituents, such as bromo or methoxy draꢀ
matically decreased the potency of the compounds. The best
compound identified was 2c with the 3ꢀ(1,3ꢀdioxolano)ꢀ2ꢀ
indolinone core and a mꢀfluoroꢀphenyl substituent. This comꢀ
pound when used in combination with KAN reduced the
MICKAN for KANꢀresistant Mtb to 2.5ꢀ5 ꢀg/mL. While CLSI
recommends MICKAN of 5 ꢀg/mL on Middlebrook 7H10 agar,
it has no recommendation for susceptibility testing by Alamar
Blue, the method used here. One study suggests a critical
MICKAN of 2.5 ꢀg/mL for Alamar Blue testing. Since our inꢀ
hibitors are able to return KANꢀresistant isolates to an MICKAN
below the critical concentration, essentially making resistant
Mtb isolate KANꢀsusceptible, such inhibitors could play a
crucial role in recovering KAN as a treatment option. Howevꢀ
er, clinical studies to support this hypothesis are yet to be unꢀ
dertaken. We are actively pursuing these avenues.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version.
Funding Sources
This work was supported by a NIH Grant AI090048 (S.G.ꢀT.), a
grant from the CCG at the U. of Michigan (S.G.ꢀT.), a grant from
the Firland Foundation (S.G.ꢀT.), and by startup funds from the
College of Pharmacy at the U. of Kentucky (S.G.ꢀT. and O.V.T.).
ACKNOWLEDGMENT
We thank Steve Vander Roest, Martha Larsen, and Paul Kirchhoff
(CCG, UM) for help with HTS.
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ABBREVIATIONS
AG, aminoglycoside; Eis, enhanced intracellular survival; HTS,
highꢀthroughput screening; KAN, kanamycin; MDR, multidrugꢀ
resistant; Mtb, Mycobacterium tuberculosis; SAR, structureꢀ
activityꢀrelationship; TB, tuberculosis; XDR, extensively drugꢀ
resistant.
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ASSOCIATED CONTENT
acetyltransferase
Eis
from
Mycobacterium
tuberculosis.
Supporting Information. The supporting information includes
the structures of scaffolds 1 and 2 (Figs. S1ꢀS3) and models of Eis
inhibitors bound to Eis (Fig. S4), experimental procedures, and
the characterization data of all new compounds synthesized and
their 1H and 13C NMR spectra (Figs. S5ꢀS56). Representative IC50
curves are also provided (Fig. S57). This material is available free
of charge via the Internet at http://pubs.acs.org.”
ChemMedChem 2012, 7, 73ꢀ77.
12. Green, K. D.; Chen, W.; Houghton, J. L.; Fridman, M.; Garneauꢀ
Tsodikova, S. Exploring the substrate promiscuity of drugꢀmodifying
enzymes for the chemoenzymatic generation of Nꢀacylated
aminoglycosides. ChemBioChem 2010, 11, 119ꢀ126.
13. Boehr, D. D.; Daigle, D. M.; Wright, G. D. Domainꢀdomain
interactions in the aminoglycoside antibiotic resistance enzyme
AAC(6')ꢀAPH(2''). Biochemistry 2004, 43, 9846ꢀ9855.
AUTHOR INFORMATION
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14. Willby, M. J.; Green, K. D.; Gajadeera, C. S.; Hou, C.; Tsodikov, O.
V.; Posey, J. E.; GarneauꢀTsodikova, S. Potent inhibitors of
acetyltransferase Eis overcome kanamycin resistance in
Mycobacterium tuberculosis. ACS Chem. Biol. 2016, 11, 1639ꢀ1646.
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Aminoglycoside acetylation no more: Inhibitors of the aminoglycoside multiacetylating enzyme Eis from Mycobacterium
tuberculosis (Mtb) were discovered and developed and were shown to restore kanamycin sensitivity of kanamycinꢀresistant
Mtb bacteria.
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O
O
O
O
ArCOCH2Br
OMe
OMe
O
N
H
N
K2CO3
11-75%
R
1a-m
O
O
O
O
ArCOCH2Br
O
O
K2CO3
34-82%
N
H
N
O
R
2a-n
R: a = Ph; b = o-F-Ph; c = m-F-Ph; d = m-Cl-Ph; e = m-Br-Ph;
f = m-OMe-Ph; g = p-F-Ph; h = p-Cl-Ph; i = p-Br-Ph; j = p-Me-Ph;
k = naphthyl; l = Et; m = t-Bu; n = m-NO2-Ph

O
O
O
O
O
OMe
O
N
N
O
R
Scaffold 1
R
Scaffold 2
Ac
Ac
Eis X
Ac
Aminoglycoside
Aminoglycoside