Dichloro-naphthoquinone as a non-classical inhibitor of the mycobacterial carbonic anhydrase Rv3588c

Article abstract of DOI:10.1039/c7md00090a

The soluble mycobacterial carbonic anhydrases Rv3588c and Rv1284 belong to a different class of carbonic anhydrases than those found in humans, making them attractive drug targets by using the inherent differences in the folds of the different classes. By screening a natural product library, we identified naphthoquinone derivatives as a novel non-classical inhibitor scaffold of mycobacterial carbonic anhydrases that lack the sulfonamide/sulfamate group and thus did not affect human carbonic anhydrase II.

Full text of DOI:10.1039/c7md00090a

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Dichloro-naphthoquinone as a non-classical inhibitor of the
mycobacterial carbonic anhydrase Rv3588c
M. A. Dallaston,a S. Rajan,a J. Chekaiban,a M. Wibowo,a M. Cross,a M. J. Coster,a R. A. Davisa,† and
A. Hofmanna,b,c,†
Received 00th January
20xx
Accepted 00th January
20xx
DOI: 10.1039/x0xx00000x
The soluble mycobacterial carbonic anhydrases Rv3588c and Carbonic anhydrases (CA) are divided into five
Rv1284 belong to a different class of carbonic anhydrases than classes, α-ζ. Human carbonic anhydrases all belong
those found in humans, making them attractive drug targets by to class α, while β-CAs are found in plants, algae,
using the inherent differences in the folds of the different classes. and bacteria. Two soluble β-CAs (Rv1284 and
By screening
a
natural product library, we identified Rv3588c) found in M. tuberculosis have been
naphthoquinone derivatives as a novel non-classical inhibitor determined as essential for the survival of the
scaffold of mycobacterial carbonic anhydrases that lack the bacterium.6,7 Their absence from the human host
sulfonamide/sulfamate group and thus did not affect human as well as significant structural differences
carbonic anhydrase II.
compared to the α-CA enzymes in mammals make
β-CAs promising targets for new therapeutic drugs.8
Carbonic anhydrases are metal-dependent enzymes
with major importance for the pH homeostasis of
organisms since they catalyse the reversible
Tuberculosis (TB) is an infectious diseases of global burden, affecting more than a third of the
world’s population with 9.6 million cases and 1.5 million deaths in 2014.1 The infection,
caused by the tubercle bacillus Mycobacterium tuberculosis, is
treated with an extended course of combination therapy (i.e.
multiple drugs taken at the same time). Many of the drugs
commonly used today have adverse side effects, including
nausea, vomiting, loss of appetite, fever, and jaundice. Current
first-line drugs used to treat TB include isoniazid, rifampin,
ethambutol, and pyrazinamide. However, resistance to these
first-line drugs is increasing, with 480,000 cases of multi-drug
resistant (MDR) TB reported worldwide in 2015.2 Second-line
drugs, such as kanamycin and fluoroquinolones, are used to
treat patients with resistant TB, but this requires a complex
treatment regimen and can cause severe side effects.3
Extensively-drug resistant (XDR) TB strains, first detected in
2006, are resistant to first- and second-line anti-tubercular
antibiotics, have occurred in 117 countries and represent
approximately 10% of MDR-TB cases.2 Thus, there is an urgent
need to develop new, effective anti-TB drugs with efficacy
against drug resistant mycobacterial strains, that account for
-
hydration of CO2 to form HCO3 and H+.
Sulfonamides and their derivatives are well-
established inhibitors of mammalian carbonic
anhydrase enzymes.9 In these “classical” carbonic
anhydrase inhibitors, which include acetazolamide, topiramate
and many others, the sulfonamide moiety (-SO2-NH2) provides
anchorage of the drug to the active site zinc ion. The
conjugated aliphatic, aromatic or heterocyclic groups engage in
interactions with amino acid residues lining the cone-shaped
active site cavity. Only few compounds that lack the
sulfonamide moiety have been identified as inhibitors of
carbonic anhydrase enzymatic activity.
In a previous investigation of a library of phenol-based natural
products against a panel of
β-carbonic anhydrases from
pathogenic organisms two compounds were identified that
displayed sub-micromolar inhibition of CA enzymatic activity.10
Moreover, both compounds showed a preference of more than
two orders of magnitude higher for fungal and mycobacterial
carbonic anhydrases (Rv1284 and Rv3588c) compared to
human members of this enzyme family. Importantly, the
exceptionally small volume of the catalytic sites of Rv1284 and
Rv3588c do not seem to allow binding of molecules with an
extent larger than substrate analogues to the dimeric
enzymes.11,12 Further support of this notion was provided by a
subsequent study revealing the redox regulation of the active
site of Rv1284.13 A range of chemical probes including the
a.Griffith Institute for Drug Discovery, Griffith University, Nathan, Queensland 4111,
Australia.
b.Faculty of Veterinary and Agricultural Sciences, The University of Melbourne,
Parkville, Victoria 3010, Australia.
c.Queensland Tropical Health Alliance, Smithfield, Queensland 4878, Australia.
† Corresponding authors.
Electronic Supplementary Information (ESI) available: Supplementary Materials and
Methods. See DOI: 10.1039/x0xx00000x
up to 20% of new TB cases in some countries.4,5
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natural product polycarpine demonstrated redox effects on apparent surface-bound location that was confirmed by site-
one of the active site cysteine residues of Rv1284 from an directed mutagenesis.
These findings suggest that larger molecules may target hit , a small library (N = 6) of N-alkylated derivatives was
sites other than the active sites to modulate the enzyme synthesised (10 15
1
-
) with starting material 1 in yields
activity of mycobacterial carbonic anhydrases. Accordingly, ranging from 27% to 88% using a previously
one can hypothesise that it is possible to inhibit these reported procedure (for details see ESI materials
enzymes with particular effectors in a non-competitive and methods).20
fashion using specific structural features absent from The effectiveness of compounds
mammalian carbonic anhydrases.
1
-
15 as
inhibitors of mycobacterial carbonic anhydrase
Here, we tested a set of compounds (N = 100) recently Rv3588c was evaluated in assays of enzymatic
added to Davis Open Access Compound Library (see below) CO2 hydration catalysed by each enzyme (see ESI
as potential non-classical inhibitors of the mycobacterial materials and methods for details). As is evident from
carbonic anhydrases Rv3588c and Rv1284 (see ESI the results listed in Table 1, 2,3-
materials and methods for details) using our established dichloronaphthoquinone (1) elicited the most
platform consisting of a thermal denaturation assay as a substantial reduction (by ~80%) of the enzymatic
first step to identify potential small-molecule ligands, activity of Rv3588c. Given the selective activity
followed by validation of hit compounds in stopped-flow of 2,3-dichloronaphthoquinone (
enzyme assays.8 From the ligand binding assay, we compounds tested in this study, the inhibitory
identified compounds based on their substantial effect of this compound on Rv3588c appears to
temperature shifts (see Table 1). Thermal denaturation be due to a specific interaction with the protein,
of 40 M Rv1284 in the absence and presence of rather than an unspecific redox reaction that
compounds was monitored using SYPRO Orange would result in an oxidised state of the enzyme.
(6.5 ); experiments were performed with a The structure-activity relationships obtained
Roche LightCycler 480 (Roche, Basel, from comparison of naphthoquinone with
Switzerland) as reported previously.13 Data were naphthalene derivatives
and 15 reveal that
analysed using the software DMAN14 and
Tm (i) halogen or single hetero-atom substituents in
1) among the
1-3
ꢀ
×
1
2-
4
6-
ꢁ
values were calculated as difference between positions 2 and 3 maximise the inhibitory
ligand and DMSO control experiments. Among the activity; (ii) extended substituents in positions 2
remaining 97 compounds, 20 showed a temperature shift and/or 3 diminish inhibitory activity. Comparison
of at least 1 K, and 76 compounds between 0 and 1 K.
Based on the identified naphthoquinone scaffold of the substituents in positions 1 and 4 are required.
synthetic compounds molecules with structural Furthermore, the absence of inhibitory effects
similarity were identified from the larger Davis Open Access observed with quinone highlight that the
Compound Library (N = 472), which led to the addition of underlying scaffold is
compounds for further testing. The majority (53%) of requirement.
compounds in this library are natural products that have In order to evaluate the specificity of the
been obtained from Australian natural sources, such as inhibitory
of compounds 1 and 9 highlights that oxygen
1-3,
5
naphthalene
a
4-8
effects
of
(1) for
2,3-
either
endophytic fungi,15 plants,16 macrofungi,17 and marine dichloronaphthoquinone
invertebrates.18 Approximately 28% of this library mycobacterial CA, we next tested the relative
constitute semi-synthetic natural product analogues,19 activity of Rv1284 in the presence of compounds
while a smaller percentage (19%) are known commercial
drugs or synthetic compounds inspired by natural products. derivatives were less effective in inhibiting
In order to determine the importance of the oxygen atoms Rv1284, with 2-methoxy-naphthoquinone (
in the initial hits, compound (2,3- showing the largest reduction of Rv1284 enzyme
1-15 (see Table 1). Clearly, the naphthoquinone
4
)
1-
3,
9
dichloronaphthalene) was sourced commercially. To further activity (by ~40%).
probe the positions of the chloro-substituents in the initial
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Figure 1: Dose-response data acquired for 2,3-
dichloronaphthoquinone (1) and N-terminally His-tagged
Rv3588c (red; IC50: 1.2 ꢀM, max. inhibition: 84%), and 2-
methoxy-naphthoquinone (4) and Rv1284 (blue; IC50: 2.3 ꢀM,
max. inhibition: 40%) at a final protein concentration of 2.5
ꢀM. Each data point represents the mean of at least three
independent measurements; error bars indicate the standard
error. The solid lines show the fits of a logistic IC50 equation
using the software SDAR.21 Figure prepared with Grace-5.1.25
(http://plasma-gate.weizmann.ac.il/Grace/) and Inkscape
Compound
DSF assay
Enzyme assay‡
Rv1284
Rv1284
Rv3588c§
human CAII
N
Relative activity N Relative activity
N
Relative activity
(%)
N
ꢁTm
(K)
(%)
(%)
1
2
3
-10.2
-10.1
-8.8
3
3
3
16.1 (4.6)
6
3
3
81.5 (4.9)
3
96.8 (0.8)
2
68.1 (9.0)
94.8 (9.9)
72.2 (14.0)
76.6 (11.5)
3
3
108.9 (9.3)
2
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4
5
-0.5
3
3
84.5 (4.7)
2
2
62.3 (6.7)
101 (10.8)
3
2
104.4 (13.1)
2
+0.1
103.7 (25.0)
6
-0.5
3
80.7 (5.2)
2
83.1 (2.5)
3
7
8
-0.4
-1.1
3
3
80.6 (4.3)
78.3 (0.9)
2
2
79.5 (5.1)
74.6 (8.4)
3
2
9
+1.3
-1.5
-2.5
3
3
3
82.8 (8.7)
98.8 (2.4)
A99.1 (2.5)
3
3
3
80.3 (4.3)
67.2 (11.5)
96.5 (1.2)
3
3
2
10
11
12
-0.7
3
93.2 (1.7)
3
110 (19.7)
2
13
14
-1.5
-0.9
3
3
103 (4.3)
110 (6.7)
3
3
103 (3.9)
79.4 (8.9)
2
2
15
-0.6
3
93.8 (3.2)
3
68.5 (8.7)
3
Table 1: Identification of hits in the natural product library and assessment of relative inhibitory activity of compounds in the CO2
hydration assay catalysed by mycobacterial carbonic anhydrases.
In the context of discovering novel anti- compounds displayed any significant effects on
mycobacterial lead molecules, any candidate human CA II (see Table 1).
molecules should be specific for mycobacterial With compounds and
1
4 emerging as the most
CAs. Therefore, the possible modulation of the active inhibitors against Rv3588c and Rv1284,
catalytic activity of human carbonic anhydrase II respectively, we finally compared the efficacy of
by the three most active compounds in this both compounds in dose-response experiments
study (
1
,
2,
4) were evaluated. None of the three against their preferred target (Figure 1). From
these results, the IC50 value of 2,3-
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dichloronaphthoquinone (
1
) with respect to activity of human CA II. Future studies should
M with a focus on structure-activity relationships of non-
maximum inhibition of 84%. In contrast, 2- carbonyl derivatives of
methoxy-naphthoquinone ( ) inhibited Rv1284
with an IC50 value of 2.3 M but at a much lower
maximum inhibition (40%). Notably, the
synthetic compound has previously been
Rv3588c was determined to be 1.2
ꢀ
1
.
4
ꢀ
Acknowledgements
1
Research in the investigators' laboratories is
funded by the Australian Research Council, the
National Health and Medical Research Council
(AH, RAD, MJC) and the Rebecca L. Cooper
Medical Research Foundation (AH). An Equity
Trustees PhD Scholarship and Australian
Government Research Training Program
Scholarship (MC) is gratefully acknowledged.
reported to exhibit antiproliferative and antifungal
activities.22 The natural product
was first isolated from
the plant Impatiens balsamina L. Several studies have
4
revealed that 4 possesses antifungal, antipruritic, and
cytotoxicity effects.23-25
Conclusions
Conflict of Interest
With the vast majority of currently known
inhibitors of carbonic anhydrases relying on the
sulfonamide moiety as an anchoring group to
the active site metal ion, the naphthoquinone
skeleton constitutes a non-classical inhibitor
scaffold with respect to CA enzymes. As
highlighted in previous studies,8,13 the crystal
The authors declare no competing interests.
Notes and references
‡
Measured in the presence of 25 ꢀM inhibitor and
structures of the β
-CAs Rv1284 and Rv3588c11
compared to protein in the absence of inhibitor; values in
brackets indicate the standard error.
N-terminally His-tagged protein.
World Health Organisation, Tuberculosis, 2016.
World Health Organisation, Multidrug-Resist. Tuberc.
MDR-TB, 2016.
reveal that there are substantial steric
constraints at the entry and exit sites of the
catalytic centres and non-linear molecules with
more than 3 or 4 atoms are virtually not able to
enter the active sites of these proteins. Such
differences in the three-dimensional structures
and topologies between carbonic anhydrases of
different classes may offer an opportunity to
design inhibitor molecules specifically targeting
non-mammalian members of this enzyme family.
In this study, we identified the natural product
§
1
2
3
4
5
6
7
8
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FDA Drug Saf. Commun., 2016.
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Dye, Lancet Lond. Engl., 2003, 362, 887–899.
C. M. Sassetti and E. J. Rubin, Proc. Natl. Acad. Sci. U. S.
A., 2003, 100, 12989–12994.
C. M. Sassetti, D. H. Boyd and E. J. Rubin, Mol. Microbiol.,
2003, 48, 77–84.
,
2,3-dichloronaphthoquinone
(1) as a non-
classical inhibitor of Rv3588c. To our knowledge,
this is the most active non-classical
mycobacterial CA inhibitor to date, with an IC50
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value of 1.2
inherent in the fold of mycobacterial CAs, we
suggest that this effector binds to a cleft on the
ꢀM. Due to the steric constraints
10 R. A. Davis, A. Hofmann, A. Osman, R. A. Hall, F. A.
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Dichloro-naphthoquinone as a non-classical inhibitor of the mycobacterial
View Article Online
carbonic anhydrase Rv3588c
DOI: 10.1039/C7MD00090A
Madeleine A. Dallaston, Siji Rajan, Janine Chekaiban, Mario Wibowo, Megan Cross, Mark J.
Coster, Rohan A. Davis & Andreas Hofmann
This study reports the most active non-sulfonamide mycobacterial CA inhibitor to date.