Biotin analogues with antibacterial activity are potent inhibitors of biotin protein ligase

Article abstract of DOI:10.1021/ml300106p

There is a desperate need to develop new antibiotic agents to combat the rise of drug-resistant bacteria, such as clinically important Staphylococcus aureus. The essential multifunctional enzyme, biotin protein ligase (BPL), is one potential drug target f

Full text of DOI:10.1021/ml300106p

Letter
pubs.acs.org/acsmedchemlett
Biotin Analogues with Antibacterial Activity Are Potent Inhibitors of
Biotin Protein Ligase
Tatiana P. Soares da Costa,†,# William Tieu,‡,# Min Y. Yap,§ Ondrej Zvarec,‡,⊥ Jan M. Bell,∥
John D. Turnidge,†,∥ John C. Wallace,† Grant W. Booker,† Matthew C. J. Wilce,*,§ Andrew D. Abell,*,‡
and Steven W. Polyak*,†
‡
†School of Molecular and Biomedical Science and School of Chemistry and Physics, University of Adelaide, South Australia 5005,
Australia
§School of Biomedical Science, Monash University, Victoria, 3800, Australia
∥Microbiology and Infectious Diseases Directorate, SA Pathology, Women's and Children's Hospital, South Australia 5006, Australia
S
* Supporting Information
ABSTRACT: There is a desperate need to develop new antibiotic agents to combat
the rise of drug-resistant bacteria, such as clinically important Staphylococcus aureus.
The essential multifunctional enzyme, biotin protein ligase (BPL), is one potential
drug target for new antibiotics. We report the synthesis and characterization of a
series of biotin analogues with activity against BPLs from S. aureus, Escherichia coli,
and Homo sapiens. Two potent inhibitors with Ki < 100 nM were identified with
antibacterial activity against a panel of clinical isolates of S. aureus (MIC 2−16 μg/
mL). Compounds with high ligand efficiency and >20-fold selectivity between the isozymes were identified and characterized.
The antibacterial mode of action was shown to be via inhibition of BPL. The bimolecular interactions between the BPL and the
inhibitors were defined by surface plasmon resonance studies and X-ray crystallography. These findings pave the way for second-
generation inhibitors and antibiotics with greater potency and selectivity.
KEYWORDS: biotin protein ligase, enzyme, enzyme inhibitor, antibiotic, medicinal chemistry
t is imperative that we identify new antibiotics to combat
drug-resistant bacteria.1 One clinically important pathogen,
Without the attached cofactor, biotin-dependent enzymes are
catalytically inactive and unable to fulfill their critical metabolic
roles. As all organisms possess between one and five biotin-
dependent enzymes, all organisms require a BPL, as there are
no alternative enzymes to perform protein biotinylation. One
important biotin-dependent enzyme is acetyl CoA carboxylase,
which catalyzes the first committed step in the fatty acid
biosynthetic pathway.4 As this pathway is essential for bacterial
cell membrane maintenance and biogenesis, they are a potential
source of new antibiotic targets in certain Gram-positive
bacteria such as S. aureus.7 S. aureus expresses a second biotin-
dependent enzyme, pyruvate carboxylase, which catalyzes the
conversion of pyruvate to oxaloacetate, thereby replenishing the
TCA cycle.8 In addition to its biotin ligase activity, certain
bacterial BPLs also function as transcriptional repressors,
making them bifunctional proteins.9 BPL recognition sites in
the S. aureus genome suggest that S. aureus BPL regulates
expression of the enzymes in the biotin biosynthesis operon, as
well as the biotin transport protein BioY.10 Thus, BPL is the
master regulator protein of all biotin-mediated events in
bacteria and, therefore, an attractive new antibiotic drug target.
In this paper, we present a novel series of biotin analogues
with potent inhibitory activity against SaBPL and antibacterial
I
Staphylococcus aureus (S. aureus), is responsible for more than
half of all life-threatening bloodstream infections. S. aureus is
challenging to control as it has the ability to rapidly acquire
drug-resistance mechanisms in both hospitals and the
community, making treatment increasingly difficult and
costly.2,3 An important strategy to combat drug resistance is
to develop novel antibiotic classes for which there are no pre-
existing resistance mechanisms. This is becoming increasingly
difficult, as most of the known chemical classes and obvious
drug targets have been well explored, leaving the more
challenging targets as a new frontier for antibacterial research.
For example, little work has been done on essential bacterial
enzymes that have mammalian paralogues due to perceived
fears of possible toxicity.4 For these targets, selective inhibition
is critical.
The ubiquitous enzyme biotin protein ligase (BPL) is one
potential antibacterial target that has not yet been compre-
hensively investigated.5 BPL is responsible for the attachment
of the cofactor biotin onto biotin-dependent enzymes. This
proceeds in two partial reactions. Initially, the adenylated
reaction intermediate biotinyl-5′-AMP is produced from biotin
and ATP. Subsequently, the biotin moiety is covalently
attached to the ε-amino group of a single target lysine residue
present in the active site of biotin-dependent enzymes.6 Here,
biotin is required to facilitate the carboxylation of metabolites.
Received: May 1, 2012
Accepted: May 23, 2012
Published: May 23, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
activity against clinical strains of S. aureus. The mode of small
molecule binding to SaBPL was defined by X-ray crystallog-
raphy and surface plasmon resonance studies. A few examples
of biotin analogues with antibacterial activity do appear in the
literature, including the natural product α-dehydrobiotin11 and
an analogue chemically modified at the N1 position required
for binding carbon dioxide (Figure 1).12 These function by first
identifying BPL inhibitors with improved ligand efficiency
(LE). Our findings provide new lead structures for further
optimization, for example, 5 and 16.
We began our search by screening analogues of biotin for
their inhibitory properties. The compounds were assayed in an
in vitro biotinylation assay measuring the incorporation of
radiolabeled biotin into protein.17 Purified recombinant BPLs
from the Gram-positive bacterium S. aureus (SaBPL18), the
Gram-negative bacterium Escherichia coli (EcBPL19), and Homo
sapiens (HsBPL20) were all assessed to investigate potency and
species selectivity (Table 1). Ki values were calculated assuming
that the mechanism of action was competitive with biotin, in
agreement with Lineweaver−Burk analysis (Supporting In-
formation, Figure S1).21 This identified biotinol (6) as a pan
inhibitor with Ki values of 3.4−4 μM for all three BPLs (Table
1). The binding mechanism was confirmed by solving the X-ray
crystal structure of SaBPL in complex with 6 (Figure 2a).
Figure 1. Chemical structures of (a) biotin 1 and its analogues (b) α-
dehydrobiotin, (c) N1 substituted biotin, and (d) the pharmacophore
investigated in this study.
being incorporated into a biotin-dependent enzyme where they
abolish catalytic activity by preventing the binding or transfer of
carbon dioxide.13−15 Importantly, these analogues are either not
specific or loose broad-spectrum antibacterial activity when
assayed in rich growth media containing biotin.11,14 Moreover,
their activity has not been linked to the inhibition of BPL as
they only transiently occupy the active site of this enzyme. A
more effective approach for antibiotic discovery requires high-
affinity inhibitors that bind directly and specifically to the
bacterial BPL target, thereby preventing all protein biotinyla-
tion. A proof of concept for this approach to antibiotic
discovery has been reported recently, targeting the BPLs from
Mycobacterium tuberculosis and S. aureus.5,16 In this current
paper, we investigated biotin analogues with a view to
Figure 2. X-ray structures of inhibitors in complex with BPL. S. aureus
BPL was crystallized in complex with biotin analogues alcohol 6 (a)
and acetylene 14 (b). The hydrogen-bonding interactions with the
inhibitors are shown.
Consistent with other BPL crystal structures that appear in the
protein database, our data revealed the biotin-binding pocket of
SaBPL to be relatively small (187 Å3) with a hydrophobic
cavity encasing the polar ureido and thiophene rings of biotin.
This suggested limited opportunity for chemical modification of
the biotin heterocycle in our inhibitor design. A narrow
a
Table 1. SAR Data for the Biotin Analogue Series
c
S. aureus
b
E. coli
Ki (μM)
H. sapiens
selectivity
b
ID
n
R
Ki (μM)
LE
LE
Ki (μM)
Hs vs Sa/Ec vs Hs/Ec vs Sa
6
7
2
3
1
2
1
2
1
2
3
OH
3.37 0.3
>20
0.52
ND
ND
ND
0.69
0.56
0.67
0.58
0.47
3.96 0.4
>20
0.51
ND
ND
ND
0.56
0.46
0.57
0.46
0.39
3.85 0.3
>20
1.1/1.0/1.2
ND
OH
3
NH2
>20
>20
>20
ND
11
16
17
5
NH2
>20
>20
>20
ND
CH3
0.05 0.01
0.52 0.06
0.08 0.01
0.3 0.05
2.40 0.02
1.10 0.12
7.30 1.9
0.90 0.1
7.33 1.0
20.0 0.3
0.14 0.02
6.43 1.4
0.2 0.03
3.50 0.5
12.0 1.83
2.8/7.9/22
12.4/1.1/14
2.5/4.5/11.3
11.7/2.1/24.4
5/1.7/8.3
CH3
CCH
CCH
CCH
14
15
a
A SAR series was investigated based upon the pharmacophore shown above. Compounds were assayed against BPLs from three species and where
b
inhibition was observed Ki values are reported. Each Ki value is the mean ( standard error) of three independent experiments. LE is ligand
c
efficiency in kcal K−1 mol−1 per heavy atom. Selectivity is expressed as ratios of Ki values. Sa is S. aureus BPL, Ec is E. coli BPL, and Hs is H. sapiens
BPL.
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a
Scheme 1
a
Conditions and reagents: (a) (i) DPPA, Et3N, THF; (ii) 6 N HCl. (b) (i) (COCl)2, DCM (ii) 2-pyrithione, 1:9 (BrCCl3:DMF), 80 °C. (c) Li-
acetylide EDA complex, DMSO, 15 °C. (d) (i) SOCl2, MeOH; (ii) LiAlH4, THF. (e) TsCl, py. (f) NaN3, DMF. (g) (i) PPh3, THF; (ii) H2O. (h)
LiBr, 2-butanone. (i) Li-acetylide EDA complex, DMSO, 15 °C. (j) LiAlH4, THF.
hydrophobic tunnel accommodates the valeric acid side chain
with the carboxyl group being solvent exposed to provide a
prime target for derivitisation. The protein:ligand interaction is
stabilized through hydrogen bonding between the hydroxyl
group (present on biotin and alcohol 6) and an NH backbone
amide of Arg 122 (Figure 2a). A series of analogues were
proposed to further explore inhibition of BPL as a route to
novel antibacterial agents. Our hypothesis was that derivatiza-
tion of the carboxyl moiety of biotin would produce tight-
binding inhibitors with increased potency and selectivity. Thus,
to establish optimal binding, modifications of biotin were
investigated that either replaced the carboxyl functionality with
motifs capable of forming direct contacts with BPL as in
compounds 3, 16, and 5, and/or modified the length of the
carbon chain (compounds 7, 11, 17, 14, and 15).
The syntheses of all of the derivatives are shown in Scheme
1. Biotin 1 and homobiotin 25 were first esterified, and the
resulting methyl esters reduced with LiAlH4 to give the alcohols
6 and 7, respectively. Tosylation followed by bromination then
gave 12 and 13, respectively, which were separately reacted
with lithium-acetylide EDA complex to give the two acetylenes
14 and 15.5 The tosylate 8 was also reacted with NaN3 to give
the azide 10, which then gave the amine 11 using Staudinger
reduction with PPh3 followed by hydrolysis of the aza-ylide
intermediate. The two tosylates 8 and 9 were also reduced with
LiAlH4 to give the alkyl derivatives 16 and 17, respectively.
Curtius rearrangement of biotin 1, on reaction with
diphenylphosphoryl azide in tert-butanol, followed by treatment
with 6 N aqueous HCl, gave amine 3.22 Finally, bromide 4 was
prepared by Barton decarboxylation of 1 using 2-pyrithione,
which on treatment with lithium-acetylide EDA complex gave
acetylene 5.5
The biotin derivatives were tested against the three BPLs
using an in vitro biotinylation assay (Table 1) to reveal potent
and selective inhibitors of SaBPL. Interestingly, increasing the
chain length of 6 by one carbon resulted in an inactive
compound (7). The crystallographic data for SaBPL bound to
6 as discussed earlier supports this observation, where the key
hydrogen bond between the hydroxyl group of 6 and the
enzyme is incompatible with the longer chain length.
Replacement of the hydroxyl group of 6 with an amine, as in
3 and 11, removed all inhibitory activity. This was somewhat
surprising given that the amine would be expected to have
similar polarity and hydrogen-bonding properties to the
hydroxyl group of 6. This observation may simply reflect the
relative pKa values of the two groups. We also assayed the
synthetic intermediate bromides (4, 12, and 13), and these
were all devoid of activity, presumably due to the steric bulk of
the halids.
We next investigated hydrophobic groups (alkyl and
acetylene) at the terminus of the carbon chain of biotin (see
5 and 14−17) that we anticipated might be accommodated by
the hydrophobic nature of the biotin pocket. Most notably 5,
14, 16, and 17 exhibited up to a 20-fold selectivity for SaBPL
over EcBPL and HsBPL. Interestingly, removal of the
tetrahydrothiophane ring in 14 removed all activity (see 18
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Figure 3. SPR analysis of BPL inhibitor binding. Surface plasmon resonance sensorgrams are shown for the binding of (a) biotin, (b) 6, (c) 16, and
(d) 5 to immobilized S. aureus BPL. Varying concentrations of the inhibitors were included in the running buffer, and analysis was performed as
described in the Supporting Information.
and 19), highlighting the importance of the fused bicyclic ring
of biotin. In the alkyl series, 16 was the most potent inhibitor
for all three enzymes (SaBPL, Ki = 0.05 μM) and displayed the
greatest LE (0.69 kcal K−1 mol−1 per heavy atom). Again, a
longer chain length (see 17) decreased potency by ≈10-fold for
the two bacterial enzymes and by 45-fold for the human
enzyme. However, significant improvement in selectivity was
apparent (see Table 1). The most potent compound in the
acetylene series was again that with the shortest carbon chain
(see 5, Ki = 0.08 μM and LE 0.67 kcal K−1 mol−1 per heavy
atom for SaBPL) with an observed decrease in potency and LE
on increasing the chain length from n = 1 to 3 (see compounds
14 and 15 in Table 1). Once again, the most selective
compound was where n = 2 (see 14; with 20- and 10-fold
selectivity for SaBPL against E. coli and H. sapiens BPL,
respectively). The acetylene 5 retained significant potency
against the target SaBPL with a Ki = 0.30 μM. The structure of
biotin acetylene (14) in complex with SaBPL was determined
to confirm both the mode of binding and our proposal that
substitution of the carboxyl group of biotin with hydrophobic
functionalities would promote binding to the target. In
agreement with the hypothesis, the complex was stabilized
through a direct hydrophobic interaction between the acetylene
and the Trp 127 (Figure 2b). Given that we observed selectivity
in this series, we propose that this interaction should be further
explored in inhibitor design. Our data strongly suggest that the
biotin pockets of the three BPLs are not as structurally
conserved as previously proposed, and this could be explored to
improve potency and selectivity.
Ideally, our antibacterial compounds should have a relatively
high affinity for BPL through slow dissociation from the
binding pocket. To study this, SaBPL was immobilized on a
sensor chip for subsequent binding studies by surface plasmon
resonance. Biotin and analogues 6, 16, and 5 were then
separately passed across the surface, providing quantitative
analysis of association and dissociation rates. The binding of
biotin to SaBPL (Figure 3a) was found to exhibit rapid on and
off rates outside the range for quantitative evaluation. Hence,
the KD (9.2 1.4 μM) was calculated using steady-state affinity.
This observation is consistent with a two-step binding
mechanism that has been reported for E. coli BPL.23 Initially,
the enzyme and biotin rapidly form a “collision complex” that is
followed by a slower conformational change to BPL that is both
rate-limiting in the binding mechanism and necessary to
stablize the enzyme−biotin complex. Alcohol analogue 6
showed similar kinetics of binding as biotin (Figure 3b, KD =
2.5
0.3 μM), representative of a dynamic equilibrium
between the free enzyme and the collision complex but unable
to overcome the rate-limiting conformational change. In
contrast, 16 and 5 displayed slower association (ka = 43.2
2.3 × 103 M−1 s−1 and 41.2 2.3 × 103 M−1 s−1, respectively)
and dissociation rates relative to both biotin and 6 (16 kd =
2.80 0.20 × 10−3 s−1 and 5 3.40 0.30 × 10−3 s−1), thereby
contributing to their significantly higher affinities (16 KD = 0.06
0.01 μM, Figure 3c, and 5 KD = 0.08 0.01, Figure 3d). The
different association and dissociation kinetics displayed by 16
and 5 as compared to biotin and 6 imply that the modes of
binding are mechanistically different. We propose that unlike
biotin, 16 and 5 do indeed induce the conformational changes
to SaBPL that are necessary to retard the inhibitors from
vacating their binding pockets. For antibacterial discovery,
inhibitors that possess high occupancy rates on their bacterial
targets are desirable.
Finally, the antibacterial activity of the most potent biotin
analogues was determined using antimicrobial growth assays.
These studies revealed significant bacteriostatic activity against
both Gram-positive and Gram-negative bacteria. Compounds
16 and 5 were the most potent against clinical isolates of S.
aureus with MICs in the range of 2−16 μg/mL, including
methicillin-susceptible and -resistant strains (Table 2). This is
consistent with the fact that these two derivatives were the most
potent inhibitors of SaBPL (Table 1). Derivative 17, containing
an extra carbon in the side chain relative to 16, was less active
against MSSA and MRSA, but interestingly, it was weakly active
against vancomycin-resistant Enterococcus (MIC 32 μg/mL).
a
Table 2. Antibacterial Susceptibility Testing
MIC50
(μg/mL)
MIC90
(μg/mL)
strain
range (μg/mL)
16
5
16
5
MSSA (n = 8)
MRSA (n = 8)
4−16
4−16
2−>64
8
8
2
8
8
4
16
16
8
16
16
8
coagulase negative
Staphylococci (n = 7)
a
MICs are shown for a library of S. aureus clinical isolates of coagulase
negative and positive strains, including methicillin-sensitive (MSSA)
and -resistant (MRSA) subtypes.
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Compounds 16 and 5 were also active against coagulase
negative Staphylococci strains. The mechanism of antibacterial
action was assessed with a complementation assay using 5 with
an E. coli K12 strain engineered to overexpress the BPL target.
Bacterial growth was monitored over 14 h in the absence or
presence of 5 at 32 μg/mL (Figure S2 in the Supporting
Information). Overexpression of EcBPL completely alleviated
antibacterial activity, strongly implying that the drug target is
indeed BPL. Finally, the toxicity of the compounds was
addressed in a mammalian cell culture model using HepG2
cells. These studies showed that the metabolic activity of the
cells was uneffected when treated with 64 μg/mL of 7, 16, 17,
5, 14, and 15.
Here, we report new data that supports the hypothesis that
BPL is a druggable antibacterial target in vitro. Our data
demonstrate that BPL inhibitors with favorable in vitro
properties also show significant antibacterial activity against
clinical isolates of methicillin-sensitive and -resistant S. aureus. It
is noteworthy that there was a positive correlation between
slow enzyme:inhibitor dissociation kinetics and potent
antibacterial activity. The quantitative data reported here help
to define the target product profile necessary for future
chemical optimization. Our biotin alkyl and acetylene series
represent new chemical scaffolds with high LE for chemical
development toward new antibacterial agents, a point that we
have begun to address as reported in our earlier publication.5
ABBREVIATIONS
■
BPL, biotin protein ligase; Ec, Escherichia coli; Hs, Homo
sapiens; LE, ligand efficiency; MIC, minimal inhibitory
concentration; Sa, Staphylococcus aureus
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ASSOCIATED CONTENT
■
S
* Supporting Information
Assay and synthetic procedures and data for selected
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Author
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*Tel: +61 3 9902 9244. E-mail matthew.wilce@monash.
edu(M.C.J.W.). Tel: +61 8 8313 5652. Fax: +61 8 8313
4358. E-mail: andrew.abell@adelaide.edu.au (A.D.A.). Tel: +61
8 8313 5289. Fax: +61 8 8313 6042. E-mail: steven.polyak@
adelaide.edu.au (S.W.P.).
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Present Address
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Funding
We acknowledge funding from the Australian Research Council
and National Health and Medical Research Council of Australia
(application number 1011806), BioInnovationSA, and the
University of Adelaide's Commercial Accelerator Scheme.
Notes
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The authors declare no competing financial interest.
M.C.J.W. is a National Health and Medical Research Council
Senior Research Fellow.
ACKNOWLEDGMENTS
■
Diffraction data were collected on the MX1 beamline at the
Australian Synchrotron, Victoria, Australia.
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