Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase

Article abstract of DOI:10.1021/ml4003277

Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.

Full text of DOI:10.1021/ml4003277

Letter
pubs.acs.org/acsmedchemlett
Fragment-Based Discovery of 6‑Azaindazoles As Inhibitors of
Bacterial DNA Ligase
Steven Howard,*,† Nader Amin,† Andrew B. Benowitz,‡ Elisabetta Chiarparin,† Haifeng Cui,*,‡
Xiaodong Deng,‡ Tom D. Heightman,† David J. Holmes,‡ Anna Hopkins,† Jianzhong Huang,‡ Qi Jin,‡
Constantine Kreatsoulas,‡ Agnes C. L. Martin,† Frances Massey,† Lynn McCloskey,‡ Paul N. Mortenson,†
Puja Pathuri,† Dominic Tisi,† and Pamela A. Williams†
†Astex Pharmaceuticals Inc., 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, United Kingdom
‡GlaxoSmithKline, Infectious Diseases TAU, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, United States
S
* Supporting Information
ABSTRACT: Herein we describe the application of fragment-
based drug design to bacterial DNA ligase. X-ray crystallography
was used to guide structure-based optimization of a fragment-
screening hit to give novel, nanomolar, AMP-competitive
inhibitors. The lead compound 13 showed antibacterial activity
across a range of pathogens. Data to demonstrate mode of action
was provided using a strain of S. aureus, engineered to overexpress
DNA ligase.
KEYWORDS: Bacterial DNA ligase, S. aureus, fragment-based drug design, structure-based optimization
acterial DNA ligase (LigA) is an NAD+-dependent
antibacterial activity, include the adenosine analogue 1 and
naphthyridine 2 (Figure 1).8,10
Benzyme, which is essential for DNA replication and has
attracted interest as a novel target for antibacterial therapy.1
LigA is responsible for ligating two strands of DNA via the
formation of a phosphodiester bond between the 3′-hydroxyl
end of one oligonucleotide and the 5′-phosphate end of
another.2,3 This process involves a three-step mechanism.
Initially, reaction between NAD+ and an active-site lysine leads
to an adenylated form of the protein. Enzyme-bound AMP is
then transferred to the 5′-phosphate end of a nicked DNA
strand. Finally, attack on the AMP-DNA bond by the 3′-
hydroxyl of a second strand of DNA seals the phosphate
backbone and releases AMP. LigA has been shown to be
essential for viability in all Gram-positive and Gram-negative
organisms tested to date.4,5 It is highly conserved across
bacterial species and is phylogenetically quite distinct from its
human, ATP-dependent, counterpart. This provides encourage-
ment that inhibitors of LigA may exhibit both broad-spectrum
antibacterial activity and selectivity over human isozymes.6
Widespread bacterial resistance to current classes of
approved antibiotics has led to an unmet medical need for
compounds with novel modes of action that are not
compromised by pre-existing resistance mechanisms.7 As a
clinically unexploited target, inhibitors of LigA would fall into
this category, and several classes of compounds have been
reported in the literature.8−11 In most cases, high throughput
screening (HTS) hits have provided chemical starting points,
which were then optimized, often using structure-based drug
design. Notable examples, shown to exhibit target-mediated
Figure 1. Inhibitors of DNA ligase described in the literature.8,10
Although in cases such as LigA molecular targeted HTS has
delivered chemical leads, success rates from antibacterial HTS
are typically lower than for targets from other therapeutic
areas.12 In this letter, we describe an alternative approach to the
discovery of LigA inhibitors using fragment-based drug design
(FBDD).13 FBDD has become established as an approach for
the generation of chemical leads for drug targets and has been
employed in a variety of therapeutic areas, including
antibacterials.14,15 In this approach specialized detection
methods are used to identify small chemical compounds,
Received: August 22, 2013
Accepted: October 10, 2013
© XXXX American Chemical Society
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Figure 2. Fragment optimization and growth of 3 toward 13. Data presented as Kd (ITC) or IC50 (biochemical assay) using DNA ligase from S.
aureus.
fragments (MW ≤ 16 heavy atoms) that bind to the drug
target, and structural biology is usually employed to establish
their binding mode and to facilitate their optimization. A key
feature of fragment-derived leads is that they are, on average,
significantly smaller and less lipophilic than historical leads
derived from HTS.16 Given that successful antibiotics are
typically associated with lower lipophilicity than other drug
classes,17,18 FBDD seems well suited to this therapeutic area.
To our knowledge, this is the first published example of a
fragment-derived LigA inhibitor.
An essential prerequisite to a successful fragment screening
cascade for LigA was the identification of a stable, deadenylated,
soakable crystal form of the protein. Constructs from multiple
pathogens, including E. faecalis, H. influenzae, S. aureus, and E.
coli, were prepared and subject to deadenylation by incubation
with nicotinamide mononucleotide (NMN). LigA constructs
showing reproducible and sustained levels of complete
deadenylation (as determined by mass spectrometry) were
progressesd to large scale crystallization trials. Of these, LigA
from S. aureus provided the most stable soakable crystal form of
the enzyme, and this was selected as the preferred system for X-
ray crystallographic screening. Approximately 1500 compounds
from our fragment library were then screened using a
combination of high throughput LigA X-ray crystallography,
ligand observed NMR (via water LOGSY), and a thermal shift
(Tm) assay. Hits were then followed up using isothermal
titration calorimetry (ITC) to determine binding affinities and
ligand efficiencies (LE).19
An additional, water-mediated, hydrogen bond exists between
the triazole nitrogen and the protein backbone NH of Ile113.
Further interaction is provided by the side chain of Tyr219,
which partially stacks with the pyrazine ring.
Pyrazine 3 offered an attractive starting point for structure-
based optimization for three reasons. First, 3 was one of the
most ligand efficient hits observed for LigA. Second, the 2-
chloro group partly fills the hydrophobic pocket (Figure 3a), a
region of the active site which is occluded in the human form of
the enzyme, providing an opportunity to obtain selectivity.20
Third, replacing the pyrazine with a pyridine would provide a
growth vector to explore the ribose-binding region, via the
pyridyl 4-position, provided this replacement was tolerated.
The pyridine analogue 4 [Kd (ITC) = 10 μM, IC50 = 17 μM,
LE = 0.54] (Figure 2) was prepared, which, gratifyingly,
showed improved activity over the fragment hit 3. One concern
was the potentially reactive chlorine at position 2. To mitigate
this risk, a number of alternative groups were explored at this
position. Deletion of the 2-substituent (compound 5) or
introduction of an electron donating substituent (e.g., OMe
analogue 6) were both detrimental to activity. 2-Alkyl
substituents (e.g., cyclobutyl 8), although appearing to fill the
hydrophobic region more effectively than chlorine, surprisingly
offered little advantage. These findings suggested that the
electronics of the 2-substituent may be more important than
sterics and that increasing the electron withdrawing nature of
the alkyl group could be advantageous. The trifluoromethyl
analogue 10 (IC50 = 16 μM, LE = 0.44) was synthesized, which
proved equipotent with 4, albeit with a modest loss in ligand
efficiency.
The starting point for chemical optimization was provided by
the fragment-based screening hit 3 [Figure 2, Kd (ITC) = 38
μM, ligand efficiency, LE = 0.50]. The X-ray crystallographic
structure of LigA (S. aureus) in complex with 3 revealed that
the fragment binds in the AMP pocket and forms hydrogen
bonds with the side chains of Lys283 and Glu110 (Figure 3a).
As described above, one of the interactions formed by these
compounds is a water-mediated hydrogen bond between the
triazole 4-position nitrogen and the backbone NH of Ile 113
(shown in Figure 3a). Our next strategy was to expand the
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ambiguous, and therefore, the exact position of the OH group
is uncertain. Although the ethanolamine motif did not improve
potency in the biochemical assay, it did result in 12 being the
first analogue to show signs of antibacterial activity, particularly
against S. aureus [MIC S.aureus (Oxford) = 8 μg/mL] (Table
1).
Table 1. MIC Data for Compounds 1, 12, and 13
MIC (μg/mL)
a
compd
1
12
13
Gram-Positive Pathogens
S. aureus Oxford
8
2
8
8
4
2
8
S. pneumoniae 1629
64
S. pyogenes 1307006P
128
Gram-Negative Pathogens
H. influenzae H128
16
128
16
E. coli 7623
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
K. pneumonia 1161486
P. aeruginosa PAO(MV)
A. baumannii BM4454
Gram-Negative Efflux Knockout Strains
H. Influenzae H128 Acr B-
E. coli 7623 TolC-
b
2
8
1
8
128
>128
>128
>128
16
16
32
32
32
>36
K. pneumonia 1161486a TolC-
P. aeruginosa PAO322
A. baumannii BM4652 (efflux mutant)
8
64
NT
NT
cytotox IC50 (μg/mL)
a
b
Compound described in ref 8. Engineered bacterial strains lacking
key efflux transporter proteins (e.g., AcrB, TolC).
Further analysis of the binding mode led us to the hypothesis
that compound 12 almost certainly binds in an unfavorable
conformation.22 This is due to both steric (hydrogens) and
electrostatic (heterocyclic nitrogen lone pairs) clashes between
the pyridine and azaindazole rings (Figure 4). This is not the
Figure 3. (a) X-ray crystal structure of fragment 3 bound to LigA (S.
aureus) showing key hydrogen bonds (purple dotted lines), a key
water molecule (red sphere), and partially resected Connoly surface.
(gray). Growth vectors toward the hydrophobic pocket, ribose binding
region, and the protein backbone are shown by the red arrows. (b) X-
ray crystal structure of 12 bound to LigA (S. aureus).
triazole ring, in order to fill the pocket more effectively, displace
this bridging water molecule and form a direct hydrogen bond
with Ile 113.
To this end, the 6-azaindazole analogue 11 (IC50 = 0.22 μM,
LE = 0.48) was prepared, which proved to be ∼70-fold more
potent than 10. An X-ray structure of 11 could not be obtained,
which we ascribed to the low solubility (solubility = 12 μg/
mL)21 of this compound. Next, attention was turned toward
exploring substitution at the pyridine 4-position in order to
access the ribose-binding pocket. The more soluble ethanol-
amine derivative 12 (solubility = 113 μg/mL) was prepared,
which was comparable in potency to 11 and enabled an X-ray
structure of the protein−ligand complex to be obtained. The
structure of 12 complexed with DNA ligase clearly shows N6 of
the 6-azaindazole making the intended hydrogen-bond to the
backbone NH of Ile113 (Figure 3b). The other interactions are
conserved. An inspection of the ribose-binding region indicates
a hydrogen-bond between the backbone carbonyl of Leu80 and
the 4-aminopyridine substituent. However, the electron density
around the last two atoms of the ethanolamine chain was
Figure 4. Illustration showing the alternative conformations of 12
(ethanolamine side chain removed for clarity).22 Electrostatic and
steric clashes are shown in red, and positive interactions are shown in
black.
case in the more favored (unbound) conformation. Replacing
the pyridine with a pyrimidine would remove the steric
hindrance in the bound conformation and also create a clash
(between nitrogen lone pairs) in the unbound conformation.
Overall, this would contribute to stabilization of the bound
state and therefore, hopefully, an increase in potency.
Consequently, pyrimidine 13 [Kd (ITC) = 0.025 μM, LE =
0.45] (Figure 2) was prepared, which proved to be 15-fold
more potent (compared to 12).
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Compound 13 exhibited antibacterial activities (Table 1),
with MIC values of 8 μg/mL or less against wild-type Gram-
positive pathogens S. aureus, S. pneumoniae, and S. pyogenes.
Moderate activities (16−32 μg/mL) were also seen against
efflux mutants of Gram-negative pathogens such as E. coli, P.
aeruginosa, and A. baumannii. The lack of activity against most
wild-type Gram-negative strains suggested that compound 13
was subject to active efflux out of the cell. This may also be
compounded with inadequate cell permeability. General
cytotoxicity against a mouse lung lymphoma cell line was not
observed for compound 13 up to the highest concentration
tested (100 μM). MIC data for literature compound 1 is shown
for comparison.8
further validation of FBDD as an effective, complementary
approach in the field of antibacterials.
ASSOCIATED CONTENT
* Supporting Information
■
S
Assay conditions (including error limits and data for reference
compounds), microbiology methods, biophysical methods, and
synthetic procedure/characterization of compounds 4−13. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(S.H.) Tel: 44-1223-226209. Fax: 44-1223-226201. E-mail:
Steven.Howard@astx.com.
■
Antibacterial activity was determined to be caused by
inhibition of LigA using a strain of S. aureus engineered to
overexpress DNA ligase (Table 2). MICs of compound 13 were
found to correlate with expression levels of DNA ligase.
*(H.C.) Tel: 1-610-917-6983. E-mail: haifeng.2.cui@gsk.com.
Notes
The authors declare no competing financial interest.
Table 2. MIC for 13 in S. aureus Parent and LigA
Overexpression Strains
a
ACKNOWLEDGMENTS
■
We would like to thank David Rees, Andrew Leach, and
Richard Jarvest for useful comments on the manuscript; and Joe
Coyle, Finn Holding, Alex Thomas, Sharna Rich, Alan Rendina,
Miriam Burman, and Emma Jones for biophysics and assay
support. Also, thanks to Nicola Wallis, Christopher Johnson,
Glyn Williams, Jeff Yon, and Christopher Murray for providing
their support during the project.
MIC (μg/mL) vs S. aureus RN4220
pYH4
pYH4-YerG (DNA ligase)
compd
13
−
+
8
−
+
8
32/16
128
a
pYH4 = vector control. pYH4-YerG = vector + DNA gene. +/− =
with or without inducer (0.1 μg/mL anhydrotetracycline).
REFERENCES
■
This result is consistent with a DNA-ligase mediated mode of
antibacterial action in this species. In the Gram-negative
pathogen E. coli, the mode of action was also tracked to LigA
inhibition by showing cross-resistance with LigA target mutants
(Table 3).23
(1) Shuman, S. J. DNA ligases: Progress and prospects. J. Biol. Chem.
2009, 284, 17365.
(2) Lehman, I. R. DNA ligase: Structure, mechanism and function.
Science 1974, 186, 790.
(3) Tomkinson, A. E.; Vijayakumar, S.; Pascal, J. M.; Ellenberger, T.
DNA ligase: Structure, reaction mechanism and function. Chem. Rev.
2006, 106, 687.
Table 3. MIC for 13 in E. coli Efflux Mutant Parent and LigA
Target Mutant Strains
(4) Streker, K.; Schafer, T.; Freiberg, C.; Brotz-Oesterhelt, H.;
̈
̈
Hacker, J.; Labischinski, H.; Ohlsen, K. In vitro and in vivo validation
of ligA and tarI as essential targets in Staphylococcus aureus. Antimicrob.
Agents Chemother. 2008, 52, 4470−4474.
MIC (μg/mL)
E. coli
TOP10 ΔTolC
compd 13
(5) Lavesa-Curto, M.; Sayer, H.; Bullard, D.; MacDonald, A.;
Wilkinson, A.; Smith, A.; Bowater, L.; Hemmings, A.; Bowater, R. P.
Characterisation of a temperature-sensitive DNA ligase from
Escherichia coli. Microbiology 2004, 150, 4171−4180.
8
TOP10 ΔTolC LigA G180E
TOP10 ΔTolC LigA R150S
TOP10 ΔTolC LigA R518H
E. coli LigA IC50 (uM)
128/64
128/64
64
(6) Swift, R. V.; Amaro, R. M. Discovery and design of DNA and
RNA ligase inhibitors in infectious microorganisms. Expert Opin. Drug
Discovry 2009, 4, 1281−1294.
0.097
(7) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.;
Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad bugs,
no drugs: No ESKAPE! An update from the Infectious Diseases
Society of America. Clin. Infect. Dis. 2009, 48, 1.
(8) Mills, S. D.; Eakin, A. E.; Buurman, E. T.; Newman, J. V.; Gao,
N.; Huynh, H.; Johnson, K. D.; Lahiri, S.; Shapiro, A. B.; Walkup, G.
K.; Yang, W.; Stokes, S. S. Novel bacterial NAD+-dependent DNA
ligase inhibitors with broad-spectrum activity and antibacterial efficacy
in vivo. Antimicrob. Agents Chemother. 2011, 1088, 55.
(9) Stokes, S. S.; Huynh, H.; Gowravaram, M.; Albert, R.; Cavero-
Tomas, M.; Chen, B.; Harang, J.; Loch, J. T., III; Lu, M.; Mullen, G. B.;
Zhao, S.; Liu, C. F.; Mills, S. D. Discovery of bacterial NAD+-
dependent DNA ligase inhibitors: optimization of antibacterial activity.
Bioorg. Med. Chem. Lett. 2011, 21, 4556.
(10) Surivet, J.-P.; Lange, R.; Hubschwerlen, C.; Keck, W.; Specklin,
J.-L.; Ritz, D.; Bur, D.; Locher, H.; Seiler, P.; Strasser, D. S.; Prade, L.;
Kohl, C.; Schmitt, C.; Chapoux, G.; Ilhan, E.; Ekambaram, N.;
Athanasiou, A.; Knezevic, A.; Sabato, D.; Chambovey, A.; Gaertner,
M.; Enderlin, M.; Boehme, M.; Sippel, V.; Wyss, P. Structure-guided
In summary, we have identified 13 (Kd = 25 nM, LE = 0.45,
LLEAT = 0.4524) as an inhibitor of bacterial DNA ligase.
Starting from pyrazine fragment 3 (Kd = 38 μM, LE = 0.50,
LLEAT = 0.56), X-ray crystallographic data was used to establish
key determinants for affinity and to guide structure based
design. In particular, a strategy of establishing additional
hydrogen bonds to the protein backbone and stabilizing the
enzyme-bound conformation led to over a 1000-fold increase in
activity, relative to starting point 3. Good LE and lipophilic
ligand efficiency (LLEAT) were maintained during this process.
Compound 13 demonstrated single-digit MICs across a range
of Gram-positive pathogens, which, in the case of S. aureus, was
shown to be target mediated. The 6-azaindaole scaffold
provides a novel, nonpurine, chemotype to the LigA field,
and to our knowledge, 13 is the first published example of a
fragment-derived LigA inhibitor. Moreover, this work provides
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Letter
design, synthesis and biological evaluation of novel DNA ligase
inhibitors with in vitro and in vivo anti-staphylococcal activity. Bioorg.
Med. Chem. Lett. 2012, 22, 6705−6711.
(11) Wang, T.; Duncan, L.; Gu, W.; O’Dowd, H.; Wei, Y.; Perola, E.;
Parsons, J.; Gross, C. H.; Moody, C. S.; Arends, R. S. J.; Charifson, P.
S. Design, synthesis and biological evaluation of potent NAD+-
dependent DNA ligase inhibitors as potential antibacterial agents. Part
II: 4-amino-pyrido[2,3-d]pyrimidin-5(8H)-ones. Bioorg. Med. Chem.
Lett. 2012, 22, 3699−3703.
(12) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L.
Drugs for bad bugs: confronting the challenges of antibacterial
discovery. Nat. Rev. Drug Discovery 2007, 6 (1), 29−40.
(13) Murray, C. W.; Rees, D. C. The rise of fragment-based drug
discovery. Nat. Chem. 2009, 1, 187−192.
(14) Eakin, A. E.; Green, O.; Hales, N.; Walkup, G. K.; Bist, S.; Singh,
A.; Mullen, G.; Bryant, J.; Embrey, K.; Gao, N.; Breeze, A.; Timms, D.;
Andrews, B.; Uria-Nickelsen, M.; Demeritt, J.; Loch, J. T., III; Hull, K.;
Blodgett, A.; Illingworth, R. N.; Prince, B.; Boriack-Sjodin, P. A.;
Hauck, S.; MacPherson, L. J.; Ni, H.; Sherer, B. Pyrrolamide DNA
gyrase inhibitors: Fragment-based NMR screening to antibacterial
agents. Antimicrob. Agents Chemother. 2012, 56 (3), 1240−1246.
(15) Hudson, S. A.; McLean, K. J.; Surade, S.; Yang, Y. Q.; Leys, D.;
Ciulli, A.; Munro, A. W.; Abell, C. Application of fragment screening
and merging to the discovery of inhibitors of the mycobacterium
tuberculosis cytochrome P450 CYP121. Angew. Chem., Int. Ed. 2012,
51, 9311−9316.
(16) Murray, C. W.; Verdonk, M. L.; Rees, D. C. Experiences of
fragment-based drug discovery. Trends Pharmacol. Sci. 2012, 33 (5),
224−232.
(17) O’Shea, R.; Moser, H. E. Physicochemical properties of
antibacterial compounds: implications for drug discovery. J. Med.
Chem. 2008, 51, 2871−2878.
(18) Manchester, J. I.; Buurman, E. T.; Bisacchi, G. S.; McLaughlin,
R. E. Molecular determinants of AcrB-mediated bacterial efflux
implications for drug discovery. J. Med. Chem. 2012, 55, 2532−2537.
(19) LE = −ΔG/HAC ≈ −RT ln(IC50)/HAC. Hopkins, A. L.;
Groom, C. R.; Alex, A. Ligand efficiency: a useful metric for lead
selection. Drug Discovery Today 2004, 9, 430−431.
(20) Han, S.; Chang, J. S.; Griffor, M. Structure of the adenylation
domain of NAD+-dependent DNA ligase from Staphylococcus aureus.
Acta Crystallogr. 2009, F65, 1078−1082.
(21) Aqueous solubility measured by a HPLC method using
ChemiLuminescent Nitrogen Detection quantification.
(22) Molecular calculations and NMR data to support this, together
with precedence from the CCD, are described in the Supporting
Information.
(23) Target mutants were generated using compound 1 (see
Supporting Information).
(24) Mortenson, P. N.; Murray, C. W. J. Comput. Aided Mol. Des.
2011, 25, 663−667.
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