A natural product inspired fragment-based approach towards the development of novel anti-bacterial agents

Article abstract of DOI:10.1039/c6md00229c

The discovery of new antibiotics with novel modes of action to combat antimicrobial resistance (AMR) is of vital importance. The natural product simocyclinone D8 (SD8) is a potent inhibitor of DNA gyrase. Its bi-functional structure and novel mode of action serve as an inspiring lead for antibiotic development. Herein we describe a proof of principle fragment-based approach towards the development of a new class of coumarin-quinolone hybrids. We demonstrate that the coumarin moiety is required for the observed inhibitory activity (IC₅₀ ～ 3 μM) of the hybrid compound, which is in part mediated through stabilisation of a cleaved-DNA intermediate.

Full text of DOI:10.1039/c6md00229c

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Hearnshaw, L. Mitchenall, P. McDermott, L. A. Howell, T. Maxwell and M. Searcey, Med. Chem. Commun.,
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A natural product inspired fragment-based approach towards the
development of novel anti-bacterial agents†
Michael J. Austin,a Stephen J. Hearnshaw,b Lesley A Mitchenall,b Paul J. McDermott,a Lesley A.
Howell,a Anthony Maxwellb* and Mark Searceya*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The discovery of new antibiotics with novel modes of action to interaction and take their inspiration from chlorofusin.4,5
combat antimicrobial resistance (AMR) is of vital importance. The Simocyclinone D8 (SD8, , Fig 1) is a further example of
1
natural product simocyclinone D8 (SD8) is a potent inhibitor of nature’s use of fragments to generate a new molecule, in this
DNA gyrase. Its bi-functional structure and novel mode of action case an antibiotic that acts as an inhibitor of DNA gyrase.6
serve as an inspiring lead for antibiotic development. Herein we DNA gyrase is a type IIA topoisomerase that regulates the
describe a proof of principle fragment-based approach towards topology of DNA.7 All topoisomerases can relax supercoiled
the development of a new class of coumarin-quinolone hybrids. DNA but DNA gyrase is unique in its ability to introduce
We demonstrate that the coumarin moiety is required for the negative supercoils at the expense of ATP hydrolysis.8 The
observed inhibitory activity (IC50 ~3 µM) of the hybrid compound, enzyme is exclusively found in bacterial cells and thus is a
which is in part mediated through stabilisation of a cleaved-DNA rational drug target. 9
intermediate.
Antimicrobial resistance and the emergence of drug-resistant
pathogens is a major health concern that threatens the society
that we live in.1 Consequently, there is an urgent need to
identify compounds with novel modes of action to treat drug-
resistant infections. Fragment-based drug design has been
utilised in the design and development of new anti-microbial
agents and is a technique based upon the use of small
molecular weight fragments, typically with low binding
affinities for their targets, that can be combined to generate
selective and high affinity molecules with therapeutic
potential.2 The main drawback to this approach is the
requirement for access to techniques such as crystallography
or high field protein NMR in order to identify the fragments.
Nature has provided a number of compounds that appear to
derive from a natural “fragment-based” approach to inhibitor
design. The MDM2/p53 inhibitor chlorofusin combines an
azaphilone and a cyclic peptide structure, neither of which
bind to their target individually but which together generated
the first natural product identified through a screen for
inhibitors for this protein-protein interaction.3 This has led to
the development of further inhibitors that target this
Figure 1. SD8 1 and constituent fragments 2 and 3. Compound 4 designed in this study
and inspired by SD8
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Figure 3. Crystal structure of SD8 (magenta) in complex with GyrA55.16 The position of
ciprofloxacin (yellow), from the S. aureus gyrase structure, is obtained by superposition
of the E. coli and S. aureus gyrase structures.14
Figure 2. Key bonding interactions for SD8, dashed lines represent hydrogen bonds,
blue lines hydrophobic interactions and blue circles water molecules.16 For clarity the
hydrogen atoms are not shown on amino acid residue
Using an SD8-inspired fragment-based approach to the design
of new DNA gyrase inhibitors, it was predicted a simple
coumarin could be tethered via a 15-Å linker to ciprofloxacin
to generate an asymmetric hybrid. The unfunctionalised
coumarin was hypothesised to still be able to form a hydrogen
bond with Arg91 present in the coumarin pocket on GyrA as
well as a hydrophobic interaction with Ser171 (Fig. 2). It was
Classical DNA gyrase inhibitors such as the fluoroquinolones,
stabilise the enzyme-DNA cleavage complex by intercalating
into double strand breaks.10 Conversely, the aminocoumarin
antibiotics inhibit the ATPase activity of the GyrB subunit.11
SD8,
1, isolated from the mycelium of Streptomyces
antibioticus Tü 6040,6 consists of an aminocoumarin (AC)
attached through an amide linkage to a rigid polyene chain,
which, in turn, is connected to an angucyclic polyketide (PK)
through a D-olivose sugar moiety. It works as a DNA gyrase
inhibitor through a novel mechanism preventing the binding of
DNA to the N-terminus of GyrA.12 DNA gyrase comprises two
subunits GyrA (97 kDa in E. coli) and GyrB (90 kDa in E. coli).
The enzyme binds to DNA as an A2B2 complex.13 DNA binds to
the N-terminal domain of GyrA and the TOPRIM domain of
GyrB comprising a ‘Gate’ segment (G-segment).14 A crystal
structure of the 59-kDa GyrA N-terminal domain complexed to
SD8 showed two binding pockets in GyrA that could
theorised that synthetic analogue
4 (Fig. 1) would be able to
adopt a favourable conformation to take advantage of both
binding sites.
The synthesis of
(Scheme 1). A modified Perkin condensation afforded the
acetate-protected 3-aminocoumarin without the need for
purification.18 Deprotection of the acetate was achieved
through heating in concentrated HCl and ethanol as
previously described.19 After cooling, the pH was adjusted to
neutral giving the free amine in a 54% yield. The mono-
functionalised product was obtained using previously
reported conditions in pyridine, with sebacoyl chloride and 7 20
The piperazine ring on commercially available ciprofloxacin
4 began from the simple salicylaldehyde 5
6
6
7
8
.
accommodate the AC and PK moieties (Fig 2 and 3).15
A
9
subsequent structure of a 55-kDa GyrA N-terminal fragment
revealed two SD8 molecules were responsible for binding to
the GyrA55 dimer and inhibiting the topoisomerase enzyme.
The AC and the PK lie in distinct binding pockets joined
through the tetraene linker, each PK spans the GyrA55 dimer
interface.16
serves as a useful functional handle for coupling. To prevent
undesired side reactions from the carboxylic acid and to
enhance the solubility of the parent compound in organic
solvents the carboxylic acid of ciprofloxacin 9 was protected as
21 Coupling of the protected ciprofloxacin fragment
an ester 10
.
using standard peptide coupling conditions provided 11 in a
SD8 itself is not drug-like due to poor solubility and cell
permeability. However, its novel mode of action serves as an
exciting lead for drug development as an example of nature’s
approach to fragment-based chemistry. Individual mono-
45% yield. Deprotection of the ester under basic conditions
gave hybrid 4 in a 40% yield.
To establish the effect of the coumarin on DNA gyrase
inhibitory activity, two controls were synthesised using the
same peptide coupling conditions (schemes 2 & 3). Aniline 12
functional fragments
M) were shown to be considerably less effective as DNA
gyrase inhibitors than the parent compound . Crucially, when
2 (Fig. 1, IC50 ~70 µM) and 3 (IC50 ~50
µ
was chosen to ensure the binding affinity of
4 was driven by
1
the ability of the coumarin to hydrogen bond to GyrA (through
the lactone). Aniline 12 was coupled with the mono-protected
sebacic acid to give 13 in 83% yield and then deprotected
used together there was no increased inhibition, signifying
that unification is vital for the observed potent activity.16
Analysis of the structure of ciprofloxacin with DNA bound to S.
aureus gyrase (PDB ref 2XCT) revealed the quinolone binding
pocket is in close proximity to the D-olivose sugar of SD8 (Fig.
3), as it has previously been shown that close sequence
similarity exists between S. aureus and E. coli gyrase.17
under basic conditions to generate the free carboxylic acid 14
.
Subsequent attachment to the ethyl ester of ciprofloxacin 10
and a final deprotection gave compound 16 in 64% yield.22
2 | J. Name., 2012, 00, 1-3
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Scheme 3. (a) EDC, monomethyl sebacate, 30% pyridine/DCM, 21 h, 65%; (b) LiOH
monohydrate, EtOH/H2O (2:1) 168 h, 51%.
Compounds 4, 6, 7, 10, 11, 15, 16, 17 and 18 were evaluated
for their ability to inhibit DNA supercoiling. DNA gyrase activity
is determined using a supercoiling assay. The individual A and
B gyrase subunits can be overexpressed, purified and mixed
together to give an active species.23 The A2B2 heterotetramer
is incubated with relaxed plasmid DNA in the presence of 1
mM of ATP. In the absence of an inhibitor, the enzyme will
supercoil the relaxed DNA, altering the linking number and
therefore changing the topological state. The supercoiled form
of plasmid DNA has a different mobility through the gel; the
condensed arrangement allows the DNA to migrate more
quickly compared to its relaxed counterpart. Thus the level of
supercoiling or lack thereof can be visually determined by
separating relaxed and supercoiled DNA, staining with
ethidium bromide and imaging under UV light. The (~4.3-Kb)
plasmid pBR322 is derived from E. coli and has become a
standard substrate for the assay.24
Scheme 1. Synthesis of asymmetric coumarin-quinolone hybrid. (a) Ac2O, NaOAc, N-
acetylglycine, reflux, 5 h, 14%; (b) conc. HCl, EtOH, Reflux, 5 h, 54%; (c) pyridine,
sebacoyl chloride, 7, reflux, 16 h, 26% (d) SOCl2, EtOH, reflux, 30 h, 60%; (e) 8, EDC, 30%
pyridine/DCM, 12 h, 45% (f) LiOH monohydrate, THF/MeOH/H2O (3:2:1), 73 h, 40%
The inhibition of DNA supercoiling by compounds 4, 16 and 18
is shown in Fig 4. The aniline control 16 still had significantly
poor inhibition at 100
µM, suggesting that it cannot make
favourable contacts in the AC binding site.
Scheme 2. Synthesis of aniline control. (a) EDC, sebacic acid monomethyl ester, 30%
pyridine/DCM, 19 h, 83%; (b) LiOH monohydrate, EtOH, H2O (2:1), 20 h, 66%; (c) 10,
EDC, 30% pyridine/DCM, 19 h, 22%; (e) LiOH monohydrate, THF/MeOH/H2O (3:2:1),
20.5 h, 64%
Conversely ciprofloxacin with an unsubstituted linker served as
a quinolone control. Coupling of the ester protected fragment
10 to monomethyl sebacate gave the penultimate compound
17 in a 65% yield. Ester hydrolysis with base gave the control
18 in a 64% yield. All final compounds had greater than 95%
purity as assessed by NMR and RP-HPLC prior to performing
biological work (supporting information).
Figure 4. Effects of Compounds, 4, 16 and 18 on DNA supercoiling by wild type E. coli
gyrase. Relaxed pBR322 plasmid DNA is used as a negative control (-), and incubated in
the presence of gyrase as a positive control (+). SD8 is used as a comparator at a
concentration of 1 µM. NC indicates nicked circle DNA; R, relaxed DNA; SC, supercoiled.
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The efficacy of ciprofloxacin
9
(IC50 0.5
µ
M) was severely Compound
M with a faint band occurring at 3
piperazine ring, with complete supercoiling observable at 25 linear band occurs at approximately the same concentration as
M for compound 18. A restoration of activity is observed inhibition in the corresponding supercoiling assay (Fig. 4). This
when the coumarin is present with an IC50 of 3 M for is consistent with observations of ciprofloxacin, which exhibits
compound , comparable to SD8 (Figure 4). These results a correlation between supercoiling and cleavage stabilisation
demonstrate a favourable interaction when fragments and inhibitory concentrations.
4
displays a strong linear band from 100
µM to 10
attenuated with the introduction of the linker onto the
µ
µ
M (Fig. 5).The loss of the
µ
µ
4
7
18 are combined that is not seen with a simple aromatic ring. These results illustrate that the coumarin fragment contributes
This is in contrast to the poor inhibition displayed by each to the inhibitory efficacy of hybrid 4. The poor inhibition
fragment individually; indicating a synergistic effect is vital for displayed by each constituent fragment of
the observed activity. The coumarins and showed no synergistic effect is vital for the observed activity. Moreover, it
appreciable effect at 50 M. Furthermore, the free amine preserves the ability to stabilise the gyrase-DNA covalent
failed to cause inhibition at physiologically irrelevant complex. The unfunctionalised scaffold will act as a suitable
concentrations of 300 M (data not shown). Of the ester template from which future structure activity relationships can
protected analogues 10 11 15 and 17, only the esterified be explored. This proof of concept work serves as an example
ciprofloxacin fragment 10 retained inhibition, albeit with of a natural product-guided fragment-based approach to
4 shows a
6
7
µ
7
µ
,
,
attenuated efficacy. Supercoiling was inhibited from 3-50
with an IC50 of ~10 M (Supplementary Information Fig. 1S).
Compounds 11 15 and 17 showed no effect at maximal
µ
M
developing novel inhibitors of DNA gyrase.
µ
,
Acknowledgements
concentrations of 100
1S).
µM (supplementary information Figure
To study the mode of action, we investigated the effect of the
compounds 16 and 18 on the gyrase cleavage-religation
MJA was funded by a Dean’s studentship. SJH was funded by
BBSRC (Grant no. BB/I002049/1). Work in AM’s lab is
supported by grant BB/J004561/1 from BBSRC and the John
Innes Foundation. We thank the EPSRC Mass spectrometry
service (Swansea) for HRMS.
4,
equilibrium (Fig 5). This would demonstrate that the
compounds were able to inhibit gyrase through the same
mechanism as ciprofloxacin. If a reaction between gyrase and
DNA in the presence of ciprofloxacin is terminated by the
addition of SDS and proteinase K, cleaved DNA is revealed. This
is a manifestation of the covalent bonds formed between the
enzyme and the DNA, which are stabilised by intercalation of
the quinolone drug into strand breaks.25 Experimentally, when
supercoiled DNA is used as the substrate, this is represented
by the presence of a linear band. SD8 was used as a negative
control, no linear band being visible due to its ability to block
DNA binding and prevent strand passage cycle taking place.
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Simocyclinone D8 served as a natural product inspiration for the synthesis of a new DNA
gyrase inhibitor.