Substituted Aminoacetamides as Novel Leads for Malaria Treatment

Article abstract of DOI:10.1002/cmdc.201900329

Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N-(3-chloro-4-fluorophenyl)-2-methyl-2-{[4-methyl-3-(morpholinosulfonyl)phenyl]amino}propanamide (compound 28) with low-nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter-screen up to 25 μm. Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for in vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification.

Full text of DOI:10.1002/cmdc.201900329

Accepted Article
Title: Substituted aminoacetamides as novel leads for malaria
Authors: Neil R. Norcross, Caroline Wilson, Beatriz Baragaña, Irene
Hallyburton, Maria Osuna-Cabello, Suzanne Norval, Jennifer
Riley, Daniel Fletcher, Robert Sinden, Michael Delves,
Andrea Ruecker, Sandra Duffy, Stephan Meister, Yevgeniya
Antonova-Koch, Benigno Crespo, Cristina de Cózar, Laura
Sanz, Francisco Javier Gamo, Vicky M. Avery, Julie A.
Frearson, David W. Gray, Alan H. Fairlamb, Elizabeth A.
Winzeler, David Waterson, Simon F. Campbell, Paul A. Willis,
Kevin D. Read, and Ian Gilbert
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To be cited as: ChemMedChem 10.1002/cmdc.201900329
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Substituted aminoacetamides as novel leads for malaria
Neil R. Norcross,1 Caroline Wilson,1 Beatriz Baragaña,1 Irene Hallyburton,1 Maria Osuna-
Cabello,1 Suzanne Norval,1 Jennifer Riley,1 Daniel Fletcher,1 Robert Sinden,2 Michael Delves,2
Andrea Ruecker,2 Sandra Duffy,3 Stephan Meister,4 Yevgeniya Antonova-Koch,4 Benigno
Crespo,5 Cristina de Cózar,5 Laura Sanz,5 Francisco Javier Gamo,5 Vicky M. Avery,3 Julie A.
Frearson,1 David W. Gray,1 Alan H. Fairlamb,1 Elizabeth A. Winzeler,4 David Waterson,6 Simon
F. Campbell,6 Paul A. Willis,6 Kevin D. Read,1* Ian H. Gilbert1*
1, Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life
Sciences, University of Dundee, Dundee, DD1 5EH.
2, Imperial College, South Kensington, London, SW7 2AZ, UK.
3, Discovery Biology, Griffith Institute for Drug Discovery, Griffith University, Nathan,
Queensland 4111, Australia.
4, Department of Pediatrics, University of California, San Diego School of Medicine, 9500
Gilman Drive 0741, La Jolla, California 92093, USA.
5, GlaxoSmithKline, Diseases of the Developing World-Tres Cantos Medicines Development
Campus, c/ Severo Ochoa, 2, Tres Cantos, 28760, Madrid, Spain.
6, Medicines for Malaria Venture, International Centre, Cointrin, Entrance G, 3rd floor Route de
Pré-Bois 20, PO Box 1826 CH-1215, Geneva 15, Switzerland.
* Authors for correspondence
1
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Graphical Abstract
Dimethyl substituent 20-fold
increase in potency
Small substituent required ortho to
sulphonamide
Phenyl analogues tolerated
Aromatic heterocycles and
cyclohexyl derivatives less
potent
Other amines tolerated
Replacement of sulphonamide with
amide or methylene not tolerated
Compound 28
Plasmodium falciparum (3D7) EC50 7 nM
HepG2 EC50 > 25 µM
Compound selection from the TCAMS library (GSK), followed by iterative rounds of inhibitor
design and synthesis afforded a single-digit nanomolar inhibitor of P.falciparum (3D7), based on
an aminoacetamide core. Compound 28 is a potent antimalarial and displayed excellent selectivity
in a mammalian counterscreen. This compound could be used as a suitable chemical tool for drug
target identification or as a lead compound for further optimisation.
2
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Abstract
In this paper we describe the optimisation of a phenotypic hit against Plasmodium falciparum,
based on an aminoacetamide scaffold. This led to a compound (28) with low nanomolar
antimalarial activity against the intraerythrocytic stages of the malaria parasite and was inactive in
a mammalian cell counter screen up to 25 µM. Inhibition of gametes in the dual gamete activation
assay suggests that this family of compounds may also have transmission blocking capabilities.
Whilst we were unable to optimise the aqueous solubility and microsomal stability to a point where
the aminoacetamides were suitable for in vivo pharmacokinetic and efficacy studies, 28 displayed
excellent antimalarial potency and selectivity and therefore, could serve as a suitable chemical tool
for drug target identification.
3
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Introduction
Malaria is a devastating parasitic disease, which is endemic in many parts of the developing world.
Human malaria is caused by five species of Plasmodium, of which, P. falciparum causes the most
number of deaths, affecting many parts of Africa. P. vivax causes severe morbidity and affects
mainly Latin America and South East Asia.[1] In 2016, there were 216 million reported cases of
malaria, and > 400,000 deaths, many of which were in children under the age of 5 years.[2] The
malaria parasite is transmitted to humans through the bite of an infected female anopheles
mosquito when taking a blood meal. Following infection, malaria parasites in the form of
sporozoites, migrate from the skin to the liver, where they invade and develop into merozoites.
When merozoites outgrow liver cells, they are released into the blood stream, invading red blood
cells, where they continue to grow and develop, repeating the cycle in the erythrocytes. During the
intraerythrocytic stages of infection, some parasites differentiate into the sexual form
(gametocytes), while others remain as the asexual form, progressing through multiple stages of
development within the blood cells. When the parasites are mature, infected red blood cells burst,
releasing parasites into the bloodstream, causing the febrile symptoms associated with malaria.
Asexual merozoites are then free to invade other red blood cells, whereas gametocytes may be
taken up through the bite of a feeding mosquito and transmitted to another human, perpetuating
the malaria parasite life cycle.[3] Current front-line therapies for the treatment of malaria are failing
due to the increasing development of drug resistance and new antimalarial treatments are urgently
needed.[4] Furthermore, novel therapies that target the malaria parasite in all life cycle stages are
required, to both treat and prevent the spread of malaria and to help in the process of eliminating
this disease.[5]
Project Initiation
To develop potential new antimalarials, we focussed our attention on the publicly available results
of a High Throughput Screening HTS campaign of nearly 2 million compounds carried out by
GlaxoSmithkline (GSK), to identify new chemical matter with antimalarial activity.[6] The
screening campaign identified >13,500 compounds malaria blood stage actives (Tres Cantos
Antimalarial Set or TCAMS). Chemical filtering and prioritization resulted in 47 different
chemotypes with antimalarial potency (EC50) < 2 µM. In this paper, we describe the selection of
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one particular series of inhibitors from the TCAMS library screen and the subsequent optimisation
of a new class of antimalarials, based on a substituted amidoacetamide core.
Hit Selection
Amidoacetamide TCMDC-123553 (Series 10)[7] was selected as a new chemical starting point
based on good antimalarial potency against chloroquine/pyrimethamine sensitive and resistant
strains, suitable physicochemical properties for further development[8] and was not subject to drug
discovery efforts from any other MMV funded programmes. Series 10 comprised a cluster of 2
compounds, TCMDC-123553 and TCMDC-125117 (Figure 1). The enamide moiety of TCMDC-
125117 was flagged as a possible Michael acceptor and with the added risk of epoxidation of the
double bond to give a reactive intermediate.[9] Therefore, our focus was directed towards TCMDC-
123553 (1) for further in vitro assessment.
Figure 1: Compounds from TCAMS Series 10.6
As a suitable chemical starting point, TCMDC-123553 (1) displayed good antimalarial activity in-
vitro and was inactive in a mammalian counter screen. In a preliminary malaria parasite rate of kill
assay (the Parasite Reduction Ratio assay[10]), 1 showed a relatively slow rate of kill, intermediate
between pyrimethamine and atovaquone (Figure 2). In this assay, parasites were treated with 10 x
EC50 concentrations of 1 and samples of parasites taken from treated cultures after 24 and 48 hrs,
and the number of viable parasites determined.
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Figure 2: Parasite Reduction Ratio (PRR) assessment of compound 1. Standard antimalarials
atovaquone, chloroquine and pyrimethamine were used as controls. Data for the controls has been
reported previously.9
Compound 1 was investigated for activity against other Plasmodium life cycle stages. In a P.
[11]
berghei liver stage schizont assay,
1 was inactive up to 50 µM. It displayed no effect in P.
cynomolgi liver stage assay[12] against both large forms and small forms (the latter indicative of P.
vivax hypnozoites) at 10 µM. Therefore, compounds of this series do not appear to be suitable for
malaria chemoprotection or radical cure. Compound 1 (incubated at a concentration of 1 µM) gave
a reduction of 99% and 81% in male and female gamete formation (sexual stages found in the
mosquito) assay, [13] respectively. However, although 1 was an inhibitor of male and female gamete
formation, it was inactive in a late stage (stage IV/V) gametocyte assay[14] at 40 µM. This latter
assay measures the ability of compounds to kill mature gametocytes. Based on the inhibition in
the dual gamete formation assay the aminoacetamide series would be expected to show
transmission blocking activity, but this would need to be confirmed in a standard membrane
feeding assay, which uses mosquito-based readouts.
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In vitro assessment of 1 showed moderate solubility (160 µM) and poor metabolic stability in
mouse liver microsomes (CLint = >50 ml/min/g liver). Therefore, our initial design efforts were
focused on improving solubility and metabolic stability, whilst improving or retaining potency. To
obtain sufficient compound exposure in vivo, our initial goal was to identify antimalarials
displaying aqueous (kinetic) solubility of ideally > 250 µM and in vitro metabolic clearance of <
5 mL/min/g liver, in mouse liver microsomes,[15] for pharmacokinetic (PK) and efficacy
experiments.
Optimisation of R1
Modifications at R1 (Figure 2 and Table 1) were initially investigated around the substituted phenyl
ring. Modifications were focussed on improving or retaining activity, whilst increasing metabolic
stability and solubility. We envisaged that an increase in metabolic stability could be achieved by
modifications to the phenyl ring by the introduction of heteroatoms to both modulate logP and
remove potential sites of metabolism. Alternatively, replacement of the phenyl ring with aliphatic
polar and/or basic groups, could simultaneously improve metabolic stability and aqueous
solubility. Removal of the chlorine atom at the C3 position to afford fluorophenyl 2, reduced
lipophilicity and improved solubility, but resulted in a small drop in potency. Removal of the
fluorine atom at C4 (4) had little effect on potency, whilst removal of both halogens (3) gave a 10-
fold reduction in activity. Furthermore, removal of one or both halogens had little effect metabolic
stability. Synthetic modifications at R1 were then directed towards aromatic heterocycles. Direct
replacement of the dihalogenated phenyl of 1 with a pyrazine (5) did not improve solubility or
metabolic stability and lost all antimalarial activity. Thiazole (6) and oxazole (7) however, did
improve solubility although all antimalarial activity was lost and metabolic stability was not
improved.
Figure 2: Developing Structure Activity Relationships (SAR)
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Table 1: Modifications at R1
Mouse
microsomal
CLint
HepG2
EC50
(µM)
Pf (3D7)
EC50 (µM)
Solb
(µM)
MWT
clogPa
R1
(ml/min/g liver)
0.14 (n = 6) c
CI 0.18-
> 25
(n = 3)
1
442
2.4
> 50
164
0.10d
0.42 (n = 4)
CI 0.52-0.34
1.45 (n = 4)
CI 1.51-1.31
0.19 (n = 4)
CI 0.28-0.13
> 25
(n = 2)
> 25
(n = 2)
> 25
2
3
4
407
389
424
2.1
2.0
2.3
45
42
> 250
183
> 50
219
(n = 2)
4.98
(n = 3)
CI 17.59-
1.64
391
5
> 50 (n = 5)
1.5
42
55
> 25
(n = 2)
> 25
(n = 2)
> 25
(n = 2)
> 25
(n = 3)
6e
7
> 50 (n = 3)
> 50 (n = 3)
>50 (n = 3)
396
380
395
383
1.6
1.3
2.2
0.8
32
9.7
3.8
37
> 250
> 250
110
8
2.67 (n = 3)
CI 3.37-2.17
9f
20
1.71 (n = 6)
CI 2.24-1.21
> 25
(n = 3)
10
11
12
431
417
406
2.2
1.7
1.2
19
10
> 250
> 250
> 250
> 25
(n = 2)
> 50 (n = 3)
> 50 (n = 3)
> 25
(n = 2)
4.6
> 25
(n = 3)
> 25
13
14
> 50 (n = 5)
> 50 (n = 3)
382
381
0.6
1.7
1.1
11
> 250
> 250
(n = 2)
(a) clogP was calculated using StarDropÒ from Optibrium. (b) Sol is kinetic solubility in water.
(c) n is the number of screening replicates. (d) CI is the 95% Confidence interval. (e) purity 84%;
(f) purity 81%.
Despite the relatively low clogP, the compounds still showed poor metabolic stability. We then
focused on aliphatic heterocycles. Substituting the R1 dihalogenated aniline of 1 for morpholine
8, lost all antimalarial activity, although led to an improvement in metabolic stability.
Cyclohexylamine 9 retained some activity, but suffered from ~ 20 fold drop in antimalarial
potency, compared to compound 1, and was metabolically unstable. The difluoro derivative (10)
8
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showed similar activity to compound 9, but with significantly improved solubility, possibly due to
the difluoro giving a small dipole moment.[16] . Replacement of the morpholine oxygen in 8 with
difluoro 11 was also inactive, and lost metabolic stability compared to 8. The addition of a nitrile
group on the cyclohexyl ring of 9, as exemplified by 12, led to a loss of activity. Replacement of
the R1 amino cyclohexyl moiety of 9 with a piperazine (13) improved metabolic stability and
aqueous solubility but not metabolic stability and also lost all antimalarial activity. Substitution at
R1 with piperidine 14 afforded a compound with reduced solubility and microsomal stability
compared to 13, and again rendered the compound inactive. Efforts to improve potency and
physicochemical properties were then directed towards changes at the R2 position.
Optimisation of R2
Substitution of the methyl group of 1 at R2 with fluorine to afford 16, displayed a small drop in
potency, but 16 was still rapidly metabolised (Table 2). Replacement of the methyl group on R2
with a chlorine atom (15), displayed a moderate improvement in potency, but aqueous solubility
and metabolic stability were still poor. Removal of the methyl group altogether with 17, did not
improve metabolic stability or solubility and gave rise to a ~ 100-fold drop in potency.
Modifications at R2 were then directed towards replacement of the morpholine sulphonamide
moiety. Removal of the morpholine oxygen atom from 1 gave the piperidine sulphonamide 18,
which showed similar potency but increased lipophilicity and decreased aqueous solubility. The
smaller and less lipophilic diethyl sulphonamide (19) and dimethyl sulphonamide (20) did not
improve metabolic stability or solubility compared to 1, although 19 retained potency, and 20 only
showed a small drop. Replacement of the sulphonamide morpholine group of 1 with a morpholine
amide (21), led to a loss of activity. However, compound 21 showed increased solubility, although
the metabolic stability was still too high. To try and recover potency, we removed the carbonyl
group of morpholine amide 21 to afford benzyl morpholine 22. We envisaged that free rotation of
the alkyl-linked morpholine around the phenyl ring of R2 may allow the morpholine group to find
an optimal interaction with the unknown biological target. However, 22 was inactive against
malaria parasites. We then explored the bridged morpholine sulphonamide moiety 23, to reduce
the planarity of the molecule, with a view to increasing solubility through the possible disruption
of crystal packing[17] Compound 23 retained only a slightly reduced potency compared to
compound 1, may have shown an marginal improvement in solubility, but was very unstable with
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microsomes. We then moved to the, bridged homomorpholino to investigate if we could modulate
activity with a larger sulphonamide. Compound 24 displayed similar potency to 23, but decreased
solubility. We then investigated changes to the linker functionality, to improve potency and
modulate physicochemical properties.
Table 2: Modifications at R2
Mouse
HepG2
Pf (3D7)
EC50 (µM)
microsomal
CLint
Solb
(µM)
R2
EC50
(µM)
MWT
clogPa
(ml/min/g liver)
0.14 (n = 6)c
CI 0.18-0.10d
> 25
(n = 3)
1
442
2.4
> 50
29
164
79
25.72
(n = 3)
CI
0.06 (n = 7)
CI 0.11-0.03
15
462
2.5
26.39-
24.69
0.58 (n = 3)
CI 0.79-0.44
> 25
(n = 2)
16
17
446
427
2.3
2.1
48
219
187
10.63 (n = 5)
CI 21.5-4.75
> 25
(n = 5)
> 50
24.03
(n = 3)
CI
24.97-
23.37
0.22 (n = 4)
CI 0.28 – 0.16
18
440
3.2
> 50
55
0.27 (n = 4)
CI 0.41-0.18
> 25
(n = 2)
19
428
400
2.2
2.3
> 50
> 50
79
0.69 (n = 4)
CI 0.79-0.59
> 25
(n = 2)
20e
181
> 25
(n = 2)
21
22
>50 (n = 3)
> 50 (n = 5)
405
392
2.3
3.2
5.6
41
> 250
> 250
> 25
(n = 3)
0.64(n =7
CI 1.0-0.35
> 25
(n = 3))
23f
454
2.7
> 50
219
0.76 (n = 3)
CI 1.1-0.55
> 25
(n = 2)
24
468
2.9
> 50
79
(a) clogP was calculated using StarDropÒ from Optibrium. (b) Sol is kinetic solubility in water.
(c) n is the number of screening replicates. (d) CI is the 95% confidence interval. Compound 17
was only weakly active with 2 of 3 replicates returning an EC50 < top concentration tested. (e)
purity 83%. (f) purity 88%.
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Increasing antimalarial activity: modifications to the linker.
Initial changes to the aliphatic linker sought to determine the importance of the linker hydrogen
bond donors for antimalarial activity, through the systematic methylation of each NH functionality.
Methylation of the R1 amide NH (25) gave rise to a loss in potency. However, methylation of the
R2 aniline NH (26) was tolerated although aqueous solubility and metabolic stability were not
improved compared with 1. Removal of the amide carbonyl to afford 27 resulted in >100 fold drop
in potency and also reduced aqueous solubility. We then investigated if the metabolic instability
of 1 was associated with metabolism a to the carbonyl – which would also be N-dealkylation, a
known Phase 1 biotransformation.[18] A common approach to reduce metabolic instability is the
geminal dimethylation of metabolically labile regions.[19] The dimethylated analogue 28 did not
show improved metabolic stability but displayed ca. 20-fold improvement in potency, compared
with hit compound 1.
Table 3: Modification of linker
Mouse
HepG2
Pf (3D7) EC50
microsomal
CLint
Solb
(µM)
Linker
EC50
(µM)
MWT
clogPa
(µM)
(ml/min/g liver)
0.14 (n = 6) c
CI 0.18-0.10d
> 25
(n = 3)
> 25
(n = 3)
> 25
(n = 2)
> 25
(n = 2)
> 25
(n = 7)
1
442
456
456
428
470
2.4
2.2
2.8
2.6
3.0
> 50
39
164
180
142
39
25
26
27
28
> 50 (n = 5)
0.29 (n = 3)
CI 0.57-0.16
22.57 (n = 3)
CI 27.83-18.76
0.007 (n = 7)
CI 0.015-0.003
> 50
37
> 50
110
(a) clogP was calculated using StarDrop from Optibrium. (b) Sol is kinetic solubility in water. (c)
n is the number of screening replicates. (d) CI is the 95% confidence interval.
Met ID studies
In order to understand what was causing the microsomal instability, we sought to identify the
chemical functional groups that were susceptible to metabolism, incubating mouse liver
microsomes in the presence 1 and monitoring for metabolites by mass spectrometry. Two main
metabolites were identified, M+H+ 458 and M+H+ 440, suggesting hydroxylation and dehydration.
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However, the exact sites of metabolism and metabolite structures were not clear from our Met ID
experiments.
Combining key SAR
A final attempt was made to investigate different combinations of previously tried functional
groups, to improve physicochemical characteristics as well as potency against P. falciparum
(Table 4). Substitution of the linker moiety with a geminal dimethyl group, combined with bridged
morpholine analogues at R2, afforded 29 and 32. Both gave a 10-fold increase in potency,
compared to 1. However, solubility and metabolic stability were not improved. New combinations
at R2, replacing the methyl group with methoxy as exemplified by 30, also resulted in a 10-fold
increase in potency and improved solubility compared to 1 although again, metabolic stability was
not improved. Substitution of the R2 morpholine on 1 with the spirocyclic morpholine
sulphonamide of 31 displayed similar potency to 1 but the desired increased levels of metabolic
stability and aqueous solubility were not obtained.
Table 4: Combining SAR
Mouse
HepG2
Pf (3D7)
EC50 (µM)
microsomal
Solb
No.
Key Compounds
EC50
(µM)
MWT
clogPa
CLint ml/min/g (µM)
liver
0.14 (n = 6) c
CI 0.18-0.10d
> 25
(n = 3)
1
442
470
2.4
3.0
> 50
> 50
164
110
0.007 (n = 7)
CI 0.015-0.003
> 25
(n = 8)
28
0.035 (n = 6)
CI 0.08-0.01
> 25
(n = 3)
29
30
496
470
3.5
3.0
> 50
40
157
219
0.014 (n = 3)
CI 0.02-0.01
> 25
(n = 2)
0.12 (n = 3)
CI 0.14-0.10
0.014 (n = 5)
CI 0.02-0.01
> 25
(n = 2)
> 25
31
32
470
482
3.0
3.3
22
167
143
> 50
(n = 3)
(a) clogP was calculated using StarDrop from Optibrium. (b) Sol is kinetic solubility in water. (c)
n is the number of screening replicates. (d) CI is the 95% confidence interval.
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Concluding remarks and future work
Through iterative rounds of drug design and synthesis, we were able to improve the antimalarial
potency of 1 by 20-fold to afford 28, which displayed single digit nanomolar potency against
Plasmodium falciparum and good cellular selectivity. It should be noted that not all compounds in
this series inhibited growth of Plasmodium falciparum to 100% in-vitro, and that there is some
evidence that some of the compounds had biphasic curves. Unfortunately, we were unable to
identify compounds that combined both potency and good metabolic stability to progress to in vivo
pharmacokinetic studies. Furthermore, we were not able to understand the reasons for the
metabolic instability. Within this series, only compounds with very low lipophilicity (clogP < 1)
displayed the desired levels of microsomal stability (< 5 mL/min/g liver). Unfortunately, these
compounds lost all antiparasitic activity. Therefore, during the optimisation of the
aminoacetamides, lowering lipophilicity was not sufficient to significantly reduce microsomal
instability to the required levels. It should also be noted that compounds with very low logP values
are likely to have a large free unbound fraction, which could lead to higher levels of clearance in
vivo.19
Given the excellent antimalarial activity and selectivity of 28, mode of action studies to determine
the molecular target of this series may be advisable, particularly given the low maximum inhibition
obtained in the in-vitro blood stage assay. These results may open new avenues to identify novel
chemotypes with improved metabolic stability, aqueous solubility and potent antimalarial activity.
Experimental
Please refer to the supplementary material for all experimental.
Author Statements
The human biological samples were sourced ethically and their research use was in accord with
the terms of the informed consents under an IRB/EC approved protocol. CC, BC, FJG and LMS
are employees of GlaxoSmithKline and own shares in the company.
Acknowledgements
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IH thanks Scottish Blood Transfusion Service for supply of expired red cells for use in anti-
parasitic assays. We acknowledge Medicines for Malaria for financial support. The University of
Dundee also acknowledges support from the Wellcome Trust (Grant 100476 and a Principal
Research Fellowship to A.H.F. Grant 079838). This work is part-funded by the MSD Scottish Life
Sciences Fund. As part of an on-going contribution to Scottish life sciences, Merck Sharp &
Dohme Limited (MSD), a global healthcare leader, has given substantial monetary funding to the
Scottish Funding Council (SFC) for distribution via the Scottish Universities Life Science Alliance
(SULSA) to develop and deliver a high quality drug discovery research and training programme.
All aspects of the programme have been geared towards attaining the highest value in terms of
scientific discovery, training and impact. The opinions expressed in this research are those of the
authors and do not represent those of MSD, nor its Affiliates.
References
[1]
E. L. Flannery, A. K. Chatterjee, E. A. Winzeler, Nature Reviews Microbiology 2014, 12,
849-862.
[2]
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