Aminopyrazinamides: Novel and specific GyrB inhibitors that kill replicating and nonreplicating mycobacterium tuberculosis

Article abstract of DOI:10.1021/cb300510w

Aminopyrazinamides originated from a high throughput screen targeting the Mycobacterium smegmatis (Msm) GyrB ATPase. This series displays chemical tractability, robust structure-activity relationship, and potent antitubercular activity. The crystal structure of Msm GyrB in complex with one of the aminopyrazinamides revealed promising attributes of specificity against other broad spectrum pathogens and selectivity against eukaryotic kinases due to novel interactions at hydrophobic pocket, unlike other known GyrB inhibitors. The aminopyrazinamides display excellent mycobacterial kill under in vitro, intracellular, and hypoxic conditions.

Full text of DOI:10.1021/cb300510w

Letters
pubs.acs.org/acschemicalbiology
Aminopyrazinamides: Novel and Specific GyrB Inhibitors that Kill
Replicating and Nonreplicating Mycobacterium tuberculosis
,∥,†
,∥,‡
§
†
†
Pravin S. Shirude,* Prashanti Madhavapeddi,* Julie A. Tucker, Kannan Murugan, Vikas Patil,
‡
†
‡
§
Halesha Basavarajappa, Anandkumar V. Raichurkar, Vaishali Humnabadkar, Syeed Hussein,
‡
‡
‡
‡
Sreevalli Sharma, V. K. Ramya, Chandan B. Narayan, Tanjore S. Balganesh,
and Vasan K. Sambandamurthy‡
†
‡
Department of Medicinal Chemistry and Department of Biosciences, AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore
5
60024, India
§
Discovery Sciences, AstraZeneca, Alderley Park, Macclesfield, U.K.
*
S Supporting Information
ABSTRACT: Aminopyrazinamides originated from a high throughput screen
targeting the Mycobacterium smegmatis (Msm) GyrB ATPase. This series displays
chemical tractability, robust structure−activity relationship, and potent antituber-
cular activity. The crystal structure of Msm GyrB in complex with one of the
aminopyrazinamides revealed promising attributes of specificity against other broad
spectrum pathogens and selectivity against eukaryotic kinases due to novel
interactions at hydrophobic pocket, unlike other known GyrB inhibitors. The
aminopyrazinamides display excellent mycobacterial kill under in vitro, intracellular,
and hypoxic conditions.
9
,10
uberculosis caused by Mycobacterium tuberculosis (Mtb) is
nolones.
This study describes the identification and
T
a leading cause of death due to an infectious agent
optimization of a novel class of aminopyrazinamides, which
display unique binding interactions at the GyrB ATPase site
resulting in selective inhibition of mycobacterium GyrB.
1
claiming 1.4 million lives annually. The emergence of
multidrug resistant tuberculosis (MDR-TB) and extremely
drug resistant tuberculosis (XDR-TB) highlights the need to
Aminopyrazinamides were identified from a high throughput
screen (HTS) of the AstraZeneca (AZ) compound collection
against Mycobacterium smegmatis (Msm) GyrB ATPase
(Supporting Information). The initial hit 1 (Table 1) displayed
moderate enzyme activity as measured by inhibition of the
target enzyme to 50% (IC50) as well as Mtb growth inhibition
as measured by minimum inhibitory concentration (MIC). We
established the fundamental structure−activity relationship
(SAR) by improving the IC50 against the target enzyme
resulting generally in potent Mtb MICs. A better understanding
of SAR would help determine if these GyrB inhibitors bind in a
similar manner to the ATPase domain as novobiocin or
2
discover drugs with novel mechanisms of action. The current
treatment for drug-susceptible tuberculosis is a four-drug
regimen administered for six months. Each of these drugs is
more than 40 years old. It is believed that, for a new TB agent
to contribute to treatment shortening, it must demonstrate an
ability to kill replicating and nonreplicating Mtb.
DNA Gyrase is a clinically validated target that catalyzes the
essential function of introducing negative supercoils in an ATP-
dependent manner. The enzyme is a heterotetrameric A2B2
protein. Negatively supercoiled DNA is crucial for cellular
processes including DNA replication, transcription, and
recombination. The druggability of DNA gyrase has been
well established through the development of the fluoroquino-
3
4
,5
6
,7
11−13
benzimidazole ureas.
were identified for the aminopyrazinamides, namely, site-1, site-
, and the hydrophobic pocket (Figure 1). The primary amide
Three diversification points for SAR
8
2
lone class of antibacterial agents. The enzyme is present in all
is an essential group at site-1, which is thought to involve a
water-mediated hydrogen bonding interaction with aspartic
acid (Asp79). The corresponding ester (2), acid (3), and
secondary amide (4) failed to display enzymatic inhibition with
prokaryotes and is the only type II topoisomerase enzyme in
Mtb. This lack of redundancy makes it an attractive target for
discovering novel drugs against tuberculosis (TB). While the
cleavage-reunion domains of GyrA and GyrB are the target of
fluoroquinolones, the GyrB ATPase domain has not been
explored extensively for anti-TB drug discovery. Hence, GyrB is
an attractive target for developing inhibitors against drug
resistant Mtb including clinical isolates resistant to fluoroqui-
Received: September 25, 2012
Accepted: December 26, 2012
Published: December 26, 2012
©
2012 American Chemical Society
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ACS Chemical Biology
Letters
Table 1. Aminopyrazinamides are Potent Inhibitors of M. smegmatis GyrB and Inhibit the Growth of Replicating M. tuberculosis
in Broth
weak or no Mtb MICs. On the basis of the known
groups at site-2 (5−16) may be involved in cation-pi
interactions with a conserved arginine residue, while a second
11−13
benzimidazole ureas,
we believe that the aryl or heteroaryl
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ACS Chemical Biology
Letters
Figure 1. Structure−activity relationship of aminopyrazinamides.
arginine residue may be involved in hydrogen bonding
interactions with either the ether O of the basic side chain or
an aryl ring N (1, 10−15). This was supported by the loss of
enzymatic and cellular potency for compound 17, which lacks
an aryl group. The basic side chain attached to the aryl or
heteroaryl ring at site-2 was found to be essential for cellular
potency, as its removal resulted in a dramatic loss of Mtb MIC
despite a sub micromolar IC50 (18). These predicted
interactions at site-1 and site-2 are conserved across known
Figure 2. M. smegmatis GyrB ATPase domain double loop-deletion
mutant (MsmGyrB-EP8) in complex with compound 6. Seen here are
site-1 interactions with Asp79 (yellow); cation-pi stacking with Arg82
and water-mediated interactions with Arg141 at site-2 (purple); and
hydrophobic pocket interactions (cyan). Figures were prepared using
PyMol (Schrodinger LLC). Final 2Fo-Fc electron density for bound
̈
compound 6 is shown contoured at 1σ. MsmGyrB-EP8 is shown as a
gray cartoon with selected side-chains in stick representation. Residues
1
1−13
GyrB inhibitors.
The substituted phenyl ring at C-6 of the
pyrazine ring is required for enzymatic activity. The
disubstituted phenyl ring shows improved potency compared
to the monosubstituted phenyl ring (5 vs 8).
5
0 to 61 have been omitted for clarity.
(
such as novobiocin), which are much broader spectrum
Conversely, the removal of the phenyl ring or introduction of
heteroaryl rings at C-6 resulted in complete loss of IC50 and
Mtb MIC (19−20) at the highest concentration tested. We
believe this novel hydrophobic interaction is unique to this
scaffold and has not been reported for other GyrB inhibitors.
Overall, the aminopyrazinamide series demonstrates a good
correlation between GyrB IC50 and Mtb MIC (Figure S1,
Supporting Information).
agents. This enzyme level specificity was further confirmed by
poor inhibition of E. coli DNA gyrase and lack of MICs against
Gram-positive and Gram-negative pathogens (Table 2).
Table 2. Aminopyrazinamides Specifically Inhibit
Mycobacterial GyrB
compound
10
11
16
novobiocin
Furthermore, to confirm the above hypothesized inter-
actions, a parallel strategy was devised to understand key
interactions at the enzyme active site with the aid of X-ray
crystallography. The crystal structure of a 22 kDa fragment of
the N-terminal domain of Msm GyrB in complex with
compound 6 confirms the two key interactions at site-1 and
site-2 (Figure 2). The primary carboxamide at site-1 makes two
hydrogen bonds with Asp79: one directly and a second via
water. The phenyl group at site-2 forms pi stacking interactions
with Arg82, while the ether O of the basic side chain makes a
water-mediated hydrogen bond with the side chain of Arg141.
The N-methyl piperazine-propoxy group at C-4 of the phenyl
ring orients toward the solvent. The crystal structure also
provided evidence of additional novel interactions involving a
hydrophobic pocket. The 3,4-dimethyl phenyl group is seen to
be oriented into a hydrophobic pocket beneath the active site
loop (Figure 2), thus explaining the SAR at this position.
Overlay of GyrB structures from other isozymes onto the
structure of Msm GyrB in complex with compound 6 suggests
this unique hydrophobic pocket may account for the specificity
observed at the enzyme level (Figure S2, Supporting
Information). This is likely due to steric interference of the
hydrophobic substituent with the amino acid residues in this
part of the active site of other GyrB isozymes. We hypothesize
the hydrophobic substituent also hinders binding to the ATP
pocket of eukaryotic kinases, thus minimizing potential off
target activity (Figure S3 and Table S1, Supporting
Information). The unique specificity of our aminopyrazinamide
series is in contrast to other known DNA gyrase inhibitors
a
Msm GyrB IC50 (μM)
0.009
1.45
0.5
<0.0025
18.65
0.5
0.022
8.37
2
0.008
0.006
2
b
Eco GyrAB IC50 (μM)
Mtb MIC (μg/mL)
Eco MIC (μg/mL)
e
>32
>32
>32
>32
>32
>4
c
Spn MIC (μg/mL)
>32
>32
>32
0.5
d
Sau MIC (μg/mL)
>32
b
0.062
c
a
Msm = Mycobacterium smegmatis. Eco = Escherichia coli. Spn =
Streptococcus pneumoniae. Sau = Staphylococcus aureus. >: end points
not determined.
d
e
The aminopyrazinamide series, while being potent ATPase
inhibitors of the Msm gyrase, are either very weakly active or
inactive against DNA gyrase from other Gram-positive and
Gram-negative enzymes, thus achieving excellent target
pathogen specificity.
Aminopyrazinamides are highly bactericidal against both
replicating and nonreplicating Mtb (Figure 3) and have potent
intracellular activity against Mtb residing within macrophages.
These compounds exhibited exposure and time-dependent
killing kinetics against Mtb grown in Middlebrook 7H9 broth
under aerobic conditions. The extent of kill reached >6 log
units by day 14 at a drug concentration of 32 μg/mL. Similarly,
a 4-log unit of kill was observed following 7 days of drug
exposure on hypoxia induced nonreplicating Mtb. The excellent
kill observed with this chemical series as compared to
novobiocin could be attributed to additional unique hydro-
phobic interactions with GyrB exploited by aminopyrazina-
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mides.
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Letters
sensitive tetrazolium dye assay. This data clearly implies that
the aminopyrazinamides are selective to mycobacteria and have
a high selectivity index against eukaryotic targets as measured
by the lack of cytotoxicity (MIC > 32 μg/mL) against C.
albicans, S. cerevisiae, and THP-1 macrophages (Table 3).
The synthesis of the aminopyrazine carboxamide (Scheme 1)
commenced with the esterification followed by diazotization of
a
Scheme 1
Figure 3. Aminopyrazinamides are highly bactericidal against M.
tuberculosis. (A) Killing kinetics of compound 11 against replicating
Mtb in 7H9 broth. (B) Cidality of aminopyrazinamides against
hypoxia induced nonreplicating Mtb. (C) Compound 11 is bactericidal
against intracellular Mtb in THP-1 macrophages.
Single-step mutants of Mtb with reduced susceptibility to
−11
pyrazinamides arose at a frequency of 2.1 × 10 when Mtb
cells were treated with 4 μg/mL of compound 11 (Table S2,
Supporting Information). Sequencing of the gyrB gene from
this resistant clone revealed a single nucleotide change in the
Mtb gyrB gene resulting in an amino acid substitution at
position 157 (Gly157 → Ser). The Gly157 → Ser mutation on
GyrB is in close vicinity to Arg180 → His or Ala92 → Ser
mutation known to confer resistance to novobiocin and
benzimidazoles, respectively, in Mtb (Figure S4, Supporting
9
,10
Information).
The representative potent compounds in the series were
profiled for preliminary DMPK (drug metabolism and
pharmacokinetics) properties (Table 3). The equilibrium
solubility for this series seems to be poor, but the physiological
relevant FassiF (fast state simulated intestinal fluid, pH 6.5)
solubility is in the range of 80 μM and 1 mM. Further, the HCl
salts of these compounds have very good equilibrium solubility.
The significant improvement of solubility (FassiF or HCl salts)
can be attributed to the basic nature of the scaffold. The protein
binding of the series was generally on the higher side with
percentage free fraction between 1 and 4. The predicted % liver
blood flow (LBF) was normally in the lower range for mouse,
human microsomes, and rat hepatocytes, respectively, and the
corresponding half-life were on higher side for microsomal
stability as compared to hepatocytes. The MIC data against
Candida albicans and Saccharomyces cerevisiae suggests that
compounds in the series are selective for bacteria and inactive
on eukaryotes. Furthermore, none of these compounds showed
any membrane disruption activity in a red blood cell hemolysis
assay (expressed as minimum lytic concentration (MLC)). We
have also observed >95% THP-1 macrophage viability
following 7 days of compound exposure at 32 μg/mL using a
a
Synthesis of aminopyrazinamides: (a) H SO (cat), MeOH, 80−90%;
2
4
(
(
b) isoamyl nitrite, CuBr , DMF, 80−90%; (c) aminophenol, 50−70%,
2
d) DIAD, polymer supported PPh , 30 min, 60−80%; (e) aryl
3
boronic acid, Pd(dppf)Cl ·DCM, CsF, MeOH, MW, 120 °C, 20 min,
6
2
0−98%; (f) 7 N ammonia in MeOH, 30 min, 60−80%; (g) t-butyl
nitrite, CuBr , DCM, 25−30%; (h) R2-OH, NaH, or K CO , DMF, 50
2
2
3
°
C, 30−50%; (i) H , Pd/C, 90−100%; (j) Pd (dba) ·CHCl ,
2
2
3
3
xanthene, Cs CO , 1,4-dioxane, 40−50%. DIAD = diisopropyl
azodicarboxylate; DCM = dichloromethane; MW = microwave;
2
3
DMF = N,N-dimethylformamide.
the amino group from commercially available 3-amino, 6-
bromopyrazine-2-carboxylic acid 21. The key intermediate
phenol 24 was prepared in excellent yield by SNAr using a
slight excess of aminophenol. The subsequent Mitsunobu
alkylation was done using polymer-supported triphenylphos-
phine, in order to ease purification, followed by a Suzuki cross-
coupling reaction under microwave conditions. The subsequent
compound 26 was treated with methanolic ammonia under
microwave conditions to afford the final compound 27. In the
Table 3. Aminopyrazinamides Exhibit Cellular Selectivity against M. tuberculosis and Preliminary DMPK Propoerties
compound
11
12
14
a
b
a
a
equillibrium solubility (μM)
10 (86) (3700)
11 (117)
6 (832)
1.93/>180
4.3/101
22/26
mouse % LBF pred (microsomes)/t1/2 (min)
human % LBF pred (microsomes)/t1/2 (min)
rat % LBF pred (hepatocytes)/t1/2 (min)
PPB (% free)
15.5/>180
7.6/77
19/25
1.0
0.63/>180
6.1/85
12.2/26
3.1
4.2
c
f
Cal MIC (μg/mL)
>32
>32
>32
d
RBC MLC (μg/mL)
>32
>32
>32
e
Sce MIC (μg/mL)
>32
>32
>32
THP-1 Macrophages at 32 μg/mL
>95% viability
>95% viability
>95% viability
a
b
c
d
e
f
FassiF. HCl salt. Cal = Candida albicans. RBC = Red blood cells. Sce = Saccharomyces cerevisiae. >: end points not determined.
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Letters
case of aminopyrimidinyl or aminopyridinyl analogues, the
basic side chains were introduced by nucleophilic substitution
reaction on substrate 30 followed by reduction to 32.
Compound 22 was subjected to a Suzuki cross-coupling
reaction under microwave conditions followed by a Buchwald
reaction using compound 32. The subsequent compound 33
was treated with methanolic ammonia to afford the final
compound 34.
and extensively drug-resistant tuberculosis: a threat to global control of
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3) Lienhardt, C., Glaziou, P., Uplekar, M., Lonnroth, K., Getahun,
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5) Koul, A., Arnoult, E., Lounis, N., Guillemont, J., and Andries, K.
2011) The challenge of new drug discovery for tuberculosis. Nature
(
(
In conclusion, we report a novel class of inhibitors,
aminopyrazinamides, which target the mycobacterial GyrB
ATPase with chemical tractability, robust SAR, and potent
antitubercular activity. In addition, these compounds have
promising attributes of specificity due to unique interactions at
a hydrophobic pocket, unlike other known GyrB inhibitors.
Compounds in the series have demonstrated excellent
mycobacterial kill under in vitro, intracellular, and hypoxic
conditions. Given the desperate need for new anti-TB agents,
we believe the aminopyrazinamide class has the potential for
further optimization to build in DMPK properties through
systematic medicinal chemistry exploration.
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METHODS
■
The experimental details can be found in the Supporting Information.
(
Franzblau, S. G., Lun, S., Guo, H., Maiga, M. C., Bishai, W. R., and
Madrid, P. B. (2012) Evaluation of gyrase B as a drug target in
Mycobacterium tuberculosis. J. Antimicrob. Chemother. 67, 415−421.
ASSOCIATED CONTENT
■
*
S
Supporting Information
(11) Charifson, P. S., Grillot, A., Grossman, T. H., Parsons, J. D.,
Details of the synthesis of all compounds, details of structure
determination, and details of biological assays is available. The
atomic coordinates and structure factors for enzyme−ligand
complex have been deposited in the protein data bank (pdb
accession code 4b6c). This material is available free of charge
via the Internet at http://pubs.acs.org.
Badia, M., Bellon, S., Deininger, D. D., Drumm, J. E., Gross, C. H.,
LeTiran, A., Liao, Y., Mani, N., Nicolau, D. P., Perola, E., Ronkin, S.,
Shannon, D., Swenson, L. L., Tang, Q., Tessier, P. R., Tian, S.,
Trudeau, M., Wang, T., Wei, Y., Zhang, H., and Stamos, D. (2008)
Novel dual-targeting benzimidazole urea inhibitors of DNA gyrase and
topoisomerase IV possessing potent antibacterial activity: intelligent
design and evolution through the judicious use of structure-guided
design and structure−activity relationships. J. Med. Chem. 51, 5243−
AUTHOR INFORMATION
■
*
5
(
263.
12) Bellon, S., Parsons, J. D., Wei, Y., Hayakawa, K., Swenson, L. L.,
Corresponding Author
Tel: + 91 080 23621212. Fax: + 91 080 23611214. E-mail:
pravin.shirude@astrazeneca.com (P.S.S.); prashanti.
madhavapeddi@astrazeneca.com (P.M.).
Charifson, P. S., Lippke, J. A., Aldape, R., and Gross, C. H. (2004)
Crystal structures of Escherichia coli topoisomerase IV ParE subunit
(24 and 43 Kilodaltons): a single residue dictates differences in
novobiocin potency against topoisomerase IV and DNA gyrase.
Antimicrob. Agents Chemother. 48, 1856−1864.
Author Contributions
These authors contributed equally to this work.
∥
(13) Grossman, T. H., Bartels, D. J., Mullin, S., Gross, C. H., Parsons,
Notes
J. D., Liao, Y., Grillot, A., Stamos, D., Olson, E. R., Charifson, P. S., and
Mani, N. (2007) Dual targeting of GyrB and ParE by a novel
aminobenzimidazole class of antibacterial compounds. Antimicrob.
Agents Chemother. 51, 657−666.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Global Alliance for
Tuberculosis (GATB). We thank the analytical support
provided by S. Rudrapatna. Our sincere thanks to the
Microbiology team for technical support in the various assays,
R. Rane for assay transfer, and R. Basu and G. Sheikh for
protein supply. We would like to thank our colleagues in the
screening, protein supply, and characterization groups in
Discovery Sciences, AstraZeneca, for their support with the
HTS. We also thank K. Das, B. Ugarkar, B. Bandodkar, S. M.
DeSousa, V. Balasubramanian, and J. Mueller for their constant
inspiration, support, and encouragement.
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