Probing the ligand preferences of the three types of bacterial pantothenate kinase

Article abstract of DOI:10.1016/j.bmc.2018.10.042

Pantothenate kinase (PanK) catalyzes the transformation of pantothenate to 4′-phosphopantothenate, the first committed step in coenzyme A biosynthesis. While numerous pantothenate antimetabolites and PanK inhibitors have been reported for bacterial type I and type II PanKs, only a few weak inhibitors are known for bacterial type III PanK enzymes. Here, a series of pantothenate analogues were synthesized using convenient synthetic methodology. The compounds were exploited as small organic probes to compare the ligand preferences of the three different types of bacterial PanK. Overall, several new inhibitors and substrates were identified for each type of PanK.

Full text of DOI:10.1016/j.bmc.2018.10.042

Accepted Manuscript
Probing the ligand preferences of the three types of bacterial pantothenate kinase
Jinming Guan, Leanne Barnard, Jeanne Cresson, Annabelle Hoegl, Justin H.
Chang, Erick Strauss, Karine Auclair
PII:
S0968-0896(18)31579-7
https://doi.org/10.1016/j.bmc.2018.10.042
BMC 14601
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
6 September 2018
23 October 2018
29 October 2018
Please cite this article as: Guan, J., Barnard, L., Cresson, J., Hoegl, A., Chang, J.H., Strauss, E., Auclair, K., Probing
the ligand preferences of the three types of bacterial pantothenate kinase, Bioorganic & Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.bmc.2018.10.042
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Graphical Abstract
Probing the ligand preferences of the three
types of bacterial pantothenate kinase
Leave this area blank for abstract info.
a
b
a
a
a
b,*
Jinming Guan , Leanne Barnard , Jeanne Cresson , Annabelle Hoegl , Justin H. Chang , Erick Strauss , Karine
a,*
Auclair
a
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A
0
B8
b
Department of Biochemistry, Stellenbosch University, Stellenbosch 7600, South Africa
Type
I
Type
II
Type
III
Pantothenate kinases

Bioorganic & Medicinal Chemistry
Probing the ligand preferences of the three types of bacterial pantothenate kinase
a
b
a
a
a
b,*
Jinming Guan , Leanne Barnard , Jeanne Cresson , Annabelle Hoegl , Justin H. Chang , Erick Strauss ,
a,
Karine Auclair
a
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 0B8
Department of Biochemistry, Stellenbosch University, Stellenbosch 7600, South Africa
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Pantothenate kinase (PanK) catalyzes the transformation of pantothenate to 4′-
phosphopantothenate, the first committed step in coenzyme A biosynthesis. While numerous
pantothenate antimetabolites and PanK inhibitors have been reported for bacterial type I and
type II PanKs, only a few weak inhibitors are known for bacterial type III PanK enzymes. Here,
a series of pantothenate analogues were synthesized using convenient synthetic methodology.
The compounds were exploited as small organic probes to compare the ligand preferences of the
three different types of bacterial PanK. Overall, several new inhibitors and substrates were
identified for each type of PanK.
Available online
Keywords:
antimetabolites
Pantothenate kinase inhibitors
PanK
2
009 Elsevier Ltd. All rights reserved.
Pantothenate kinase
Coenzyme A
1
. Introduction
Antibacterial resistance is growing to become a severe health
largely depends on the PanK type found in the bacterium. For
bacteria harboring the promiscuous type I PanK, such as E. coli,
pantothenamides are believed to impede growth after being
1
threat in all parts of the world. To address this global crisis,
there is an urgent need to validate new antibacterial targets and
develop novel antibacterial agents. Coenzyme A (CoA) is an
essential cofactor for all living organisms. It participates in over
1
tricarboxylic acid cycle, lipid biosynthesis and catabolism, amino
acid biosynthesis, and as carrier of the acetyl group in a range of
acetylation reactions. Pantothenate (Figure 1), also known as
vitamin B , is the precursor for CoA biosynthesis. Its ATP-
dependent phosphorylation to produce 4′-phosphopantothenate is
catalyzed by pantothenate kinase (PanK), and is the first and
committed step of the universal CoA biosynthetic pathway. This
is followed by four additional steps to finally produce CoA.
The CoA biosynthetic steps are conserved across all kingdoms,
but the structure and mechanism of the enzymes involved vary
significantly. The utilization of pantothenate has therefore been
suggested as a novel target for the development of antimicrobials
transformed by the CoA biosynthetic enzymes into the
9–13
corresponding CoA antimetabolites,
which subsequently may
2
interfere with a series of essential CoA-related pathways. In S.
aureus, the only bacterium known to express a type II PanK, the
00 different reactions in central metabolic processes such as the
pantothenamides have been shown to act through different modes
9,14–18
of action.
Apart from being transformed into inhibitory CoA
antimetabolites, it has been demonstrated that the
pantothenamides’ inhibitory effect also results from the
pantothenamides being trapped on the S. aureus PanK after their
3
,4
5
transformation into the corresponding 4′-
9,16–18
phosphopantothenamides.
Finally, pantothenamides have no
5
effect on bacteria that have only a type III PanK such as
Pseudomonas spp., Clostridium spp., Neisseria spp., or
Helicobacter spp., because the amide substituent is not
accommodated in the tighter pantothenate binding sites of most
type III PanKs. In fact, the binding sites of type III PanKs are
very specific, and to date these enzymes have been shown to
accept only pantothenate as a substrate. It is worth noting that
although most bacteria carry only one type of PanK, some
bacteria harbor genes for two different types of PanK; for
example, some mycobacteria and bacilli have genes coding for
3
–5
6
–7
with low cytotoxicity.
Since the discovery of pantothenate by Williams et al. in
8
1
933, many pantothenate antimetabolites have been reported to
inhibit the growth of Escherichia coli and Staphylococcus
aureus. Of these, the pantothenamides are arguably the most
studied. It has been demonstrated that their mode of action
1
9
7
both a type I and a type III PanK.
6
–7
—
——

Corresponding authors. karine.auclair@mcgill.ca; estrauss@sun.ac.za

alanine moiety. These modifications allowed not only the
identification of novel inhibitors and substrates, but also revealed
important differences in the binding preferences of the three
types of PanK. This information should provide guidance in the
design of ligands that selectively target a specific PanK type.
2
. Results and discussion
2
.1. Synthesis
Pantothenate analogues 1a–j that were modified at the
primary hydroxyl group, and 1k–l that were modified at the
secondary hydroxyl group, were prepared from intermediates 2a–
l via benzyl deprotection in the presence of Pd(OH) /C (Scheme
2
1
). For the synthesis of the intermediates, D-pantothenate was
used as a starting material in all cases, except in the case of 2i
where D-pantolactone was used (Scheme 2).
Figure 1. Pantothenic acid, showing the four sites where
modifications were introduced to produce the molecules
presented here.
Intermediates 2a–f were prepared as follows: the two
hydroxyl groups of pantothenate were each protected with a TBS
group (Scheme 2A), before protection of the carboxylate as a
benzyl ester to generate compound 3. Next, 1.1 equivalent of
PPTS was used to selectively deprotect the TBS group on the
primary hydroxyl group and yield compound 4. Subsequent
oxidation with DMP produced aldehyde 5. To synthesize the
amines 2a–f, aldehyde 5 was condensed with the desired amine
Apart from pantothenate antimetabolites, several inhibitors
9
,18,20–22
have also been reported for type I and II PanKs.
contrast, only one inhibitor of a type III PanK has been reported:
an ATP mimic with a K of 164 ± 3 M (a 3-fold improvement
In
i
relative to the K of ATP which is 510 M) for the inhibition of
m
2
3
Bacillus anthracis PanK, however, no bacterial growth
inhibition was reported for this compound.
before NaBH reduction to yield compounds 6a–f. Compounds
4
2
a–f were isolated after TBAF-mediated TBS-deprotection.
In light of this background, it is clear that the design of novel
pantothenate antimetabolites and pantothenate-related PanK
inhibitors can benefit from a better understanding of the ligand
preferences of all three types of bacterial PanK. We report here
new pantothenate analogues and their use as probes to study the
specificity of the pantothenate binding pockets of the type I, II
and III PanKs. The study of ATP binding pockets was not
pursued, mainly because type III PanKs show poor affinity for
Similarly, 2g was also generated via reductive amination of
aldehyde 5, but this was followed with ring opening of diketene
in the presence of 2-hydroxylpyridine, and full deprotection
using TFA (Scheme 2B). To synthesize the carboxylate 2h
(Scheme 2C), aldehyde 5 was oxidized to carboxylic acid 7 by
Pinnick oxidation, followed by TBS deprotection using TBAF.
Synthesis of 2i (Scheme 2D) started by combining D-
pantolactone and the 4-toluenesulfonate salt of β-alanine benzyl
ester in the presence of diethylamine at 60°C, to produce
compound 8. This compound was next used as a nucleophile to
open the ring of diketene and generate 2i. Compounds 2j–l were
prepared from compound 3 (Scheme 2E). Both TBS groups were
first removed using CsF, generating the negatively charged
alkoxides, to which methyl iodide was added in situ to form 2j–l
in one pot.
ATP (with K values in the millimolar range), and because of the
m
concern of generating non-specific inhibitors that may also
inhibit many other ATP-dependent enzymes.
The pantothenate analogues presented here can be categorized
into four classes, depending on where the modification is located
(
Figure 1): 1) the primary hydroxyl moiety; 2) the geminal-
dimethyl group; 3) the secondary hydroxyl group; or 4) the -
Scheme 1. Synthesis of compounds 1a–l.

a
Scheme 2. Synthesis of compounds 2a–l. R is the same as in Scheme 1, except for compound 6e, in which R is
.
1
1
TBSCl: tert-butyldimethylsilyl chloride; DCC: dicyclohexylcarbodiimide; DMAP: 4-(dimethylamino)pyridine; PPTS: pyridium p-
toluenesulfonate; DMP: Dess-Martin periodinane; TBAF: tetrabutylammonium fluoride; TFA: trifluoroacetic acid; DEA: diethylamine;
DIPEA: N,N-diisopropylethylamine.
Compounds modified at the -alanine moiety (9a–k) or at the
geminal-dimethyl group (9l) were synthesized as shown in
Scheme 3. Methyl esters 9a–c and 10 were prepared directly
from the relevant lactones via ring-opening with the desired
amine. Hydrolysis of 9a,c and 10 separately with lithium
hydroxide quickly generated compounds 9d,e and 9l
aeruginosa pantothenate kinase (PaPanK , i.e. a type III PanK)
were selected as representatives to study these PanK types. S.
III
aureus pantothenate kinase (SaPanK ) is the only known
II
bacterial type II PanK, and was used here to represent this type of
PanK.
We first tested all compounds that cannot act as PanK
substrates because they either lack the primary hydroxyl that is
phosphorylated by PanK (as is the case for compounds 1a–d,f–
h), or this hydroxyl is blocked (as seen for 1i–k). These
compounds were screened at a fixed concentration of 200 M to
determine their ability to inhibit the activity PanK enzymes
acting on 25 µM (for EcPanK and SaPanK ) or 30 µM (for
respectively. Compounds 9f–k were designed to mimic known
pantothenamides, with the triazole ring acting as a bioisostere of
the amide group that is susceptible to hydrolysis by enzymes
2
4
present in the serum of the host. Specifically, 9f and 9g were
designed as analogues of N-pentyl and N-phenethyl α-
pantothenamide respectively, 9h as an analogue of N-butyl
pantothenamide (N4-Pan), 9i and 9j as two regioisomeric
analogues of N-pentyl pantothenamide (N5-Pan), and 9k as an
I
II
PaPanK ) pantothenate. As shown in Figure 2, compound 1g
III
9
exhibits weak inhibition of all three PanK types. We propose that
the acetoacetamide moiety of 1g may mimic the polar
diphosphate group of ATP in the ternary complex or the
transition state. The acetoacetate group has previously been
analogue of N-propyl homopantothenamide. They were
2
5–26
synthesized as reported elsewhere.
2
.2. Structure-activity relationship (SAR) studies for
2
7
differentiated inhibition of PanK types
shown to mimic diphosphate moieties. For EcPanKI,
compounds 1c,f,j also show some inhibition, and 1a to a lesser
extent, suggesting that this enzyme can tolerate either N or O at
the primary alcohol position. In contrast, PaPanKIII seemed to
For all enzyme activity and inhibition studies, E. coli
pantothenate kinase (EcPanK , i.e. a type I PanK), and P.
I

a
Scheme 3. Synthesis of compounds 9a–l. Alternatively: 115°C for 2 hours in a microwave reactor.
accommodate an amino group at this carbon better than ethers
(e.g. 1a vs 1j). Interestingly, the diacid 1h shows inhibition only
for SaPanK , even though the crystal structure of SaPanK bound
II
II
to 4′-phospho-N5-Pan (PDB: 4M7Y) does not show any residues
in the vicinity of the introduced carboxylate that would likely
1
7
form stabilizing interactions with this group.
When compounds 1e,l and 9a–l were studied as potential
inhibitors of the PanK enzymes, none of them showed any
inhibition at ≥100 M. These compounds were also characterized
for their ability to act as substrates of the respective PanKs (vide
infra).
2
.3. SAR studies for PanK types showing differentiated activity
All the compounds containing a primary alcohol at (1l, 9a–l)
Figure 2. Activity of the respective PanK enzymes acting on 25
or near C-4′ (1e) were next tested for their ability to act as
substrates of the three PanKs. In those cases where activity was
observed, the relevant kinetic parameters were determined and
the results are summarized in Table 1.
µM (for EcPanK and SaPanK ) or 30 µM (for PaPanK )
I
II
III
pantothenate in the presence of 200 µM of the indicated
compounds 1a–d,f–k. The activity is reported relative to that of a
control reaction without inhibitor, which was set at 100%.

a
Table 1. Kinetic parameters of the PanK enzymes acting on compounds 1e,l, 9a–l as substrates.
Ec PanKI
Sa PanKII
Pa PanKIII
kcat
-1
Km
kcat
kcat/Km
-1 -1
Km
kcat
-1
(s )
kcat/Km
-1 -1
(mM .s )
Km
kcat/Km
-1 -1
(mM .s )
20 ± 5
-
1
(M)
17 ± 1
(s )
1.29 ± 0.02
(mM .s )
76 ± 5
26
(M)
19 ± 1
(M)
20 ± 5
(s )
Pantothenate
N5-Pan
2.16 ± 0.05 115 ± 10
9
0.44 ± 0.03
0.39 ± 0.01
2
6
26
9
9
36 ± 3
0.93 ± 0.04
26 ± 2
<1.5
>290
1
1
9
9
e
l
a
b
16 ± 3
156 ± 26
292 ± 65
0.49 ± 0.02
0.91 ± 0.04
0.80 ± 0.06
31 ± 6
5.8 ± 1.0
2.7 ± 0.6
36 ± 2
ns
4.0 ± 0.1
115 ± 12
b
ns
ns
8.3 ± 0.2
7.1 ± 0.2
b
9
9
9
9
9
9
9
9
9
9
c
d
e
f
ns
62 ± 12
1.59 ± 0.07
26 ± 5
60 ± 2
64 ± 5
126 ± 29
3.4 ± 0.1
3.4 ± 0.1
4.7 ± 0.5
57 ± 3
54 ± 6
683 ± 72 0.98 ± 0.04 1.4 ± 0.2
2
6
26
26
190 ± 20
0.96 ± 0.04
1.09 ± 0.02
1.08 ± 0.04
0.72 ± 0.01
1.11 ± 0.06
5.1 ± 0.6
g
h
i
25 ± 3
50 ± 8
33 ± 5
21 ± 5
44 ± 5
22 ± 4
22 ± 3
53 ± 13
37 ± 12
115 ± 9
6.8 ± 0.7 0.73 ± 0.04 107 ± 16
7.1 ± 0.4 0.82 ± 0.02
j
8.5 ± 1.2 0.82 ± 0.05
369 ± 381 7.6 ± 4.3
97 ± 19
21 ± 33
2
6
26
26
k
l
1000 ± 200
0.49 ± 0.06
0.49 ± 0.11
64 ± 14 0.32 ± 0.02 5.0 ± 1.1
a
Blank cells mean that the particular PanK did not show activity toward the indicated compound under the tested conditions;
b
value of k /K estimated from the linear slope (v = k /K ·[E]·[S] when [S] << K );
cat
0.5
cat
0.5
0.5
ns: not saturated at 200 M.
Compound 1e contains a primary hydroxyl group which is
shifted four atoms away from that of the natural substrate. It was
not found to act as a substrate for any of the PanK tested. On the
other hand, when the secondary hydroxyl group of pantothenate
is blocked with a methyl substituent, as in the case of 1l, the
molecule was only accepted as an EcPanK substrate, with a K
between the terminal carboxylate group of pantothenate and Thr-
180′, Tyr-92, Arg-102 and Gly-99 residues (Figure 3C)
contribute a large proportion of the affinity. For EcPanK , both
I
one or two carbon-linkers between the amide and the carboxylate
are well tolerated, as demonstrated with compounds 9a,b,d and
9f–j. These are all good EcPanK substrates, a finding that is in
I
m
I
9
value similar to that of pantothenate. This suggests that
agreement with previous studies of pantothenamide analogues.
methylation does not significantly disrupt the interaction between
In contrast, the compounds containing a three-carbon-linker
this hydroxyl group and His-177 of EcPanK (Figure 3A), and
(9c,e,k) are poorly or not accepted as substrates by EcPanK , in
I
I
2
8,30–31
that there is space for modifications near this secondary hydroxyl
agreement with earlier studies of 9e.
This suggests that
2
8
group. The crystal structure of SaPanK bound to 4′-phospho-
regardless of the identity of the terminal group (whether a methyl
ester in 9a–c, a carboxylate in 9d–e, or a substituted triazole in
9f–k), the linker length is determinant for binding affinity to
II
N5-Pan (Figure 3B) does not indicate any interactions between
the secondary hydroxyl group and the enzyme, suggesting that
the lack of activity is likely due to steric hindrance. In PaPanKIII,
the secondary hydroxyl group of pantothenate forms two
EcPanK . The tolerance of this enzyme for methyl esters such as
I
9a,b can be rationalized in light of the crystal structure of the
2
9
hydrogen bonds with the carboxylate of Asp-101. In this case,
methylation likely disrupted at least one of these interactions,
and/or cannot be accommodated in the tight active site, leading to
no activity being observed for 1l. Overall, these differences in
how the PanK enzymes interact with the secondary hydroxyl
group of the pantothenoyl moiety suggests that modifications at
this position may allow for the development of pantothenate
antimetabolites or inhibitors that are selective for EcPanKI.
pantothenate–EcPanK complex (PDB: 1SQ5), which shows that
I
only one oxygen in the carboxylate group of pantothenate is
involved in hydrogen bonds with the side chains of residues Tyr-
2
8
240 and Asn-282 (Figure 3A). In contrast, the crystal structure
of the pantothenate–PaPanKIII complex (PDB: 2F9W) suggests
that both oxygen atoms of the carboxylate group of pantothenate
2
9
participate in hydrogen bonds with the protein (Figure 3C). In
the case of SaPanK , the enzyme was found to tolerate substrates
II
with a one- (9a,f,g), two- (9b,h–j), or three-carbon-linker
When modifications were introduced at the -alanine end of
the molecule (e.g. 9a–k), both EcPanK and SaPanK accepted
30–32
(9c,e,k), in agreement with previous reports for 9e.
The only
I
II
exception is the free acid 9d, with a one-carbon-linker; however,
its analogous methyl ester (9a) is accepted, indicating that for the
shorter compounds ionization interferes with binding.
most of these molecules as substrates, while PaPanKIII did not.
This was expected based on results with pantothenamides which
are often tolerated by EcPanK and SaPanK , but not by
I
II
9
PaPanK . Only the carboxylate 9e was found to act as a
Several important features of the SaPanK pantothenate
III
II
substrate for PaPanK , which might be explained by the
flexibility of the three-carbon linker present in compound 9e
binding site were recently discovered in a SAR study of
III
1
7
pantothenamides. This study defined a binding pocket in which
the N-substituent of these compounds are accommodated, and in
which several hydrophobic, π-stacking and hydrogen bonding
interactions are possible. Apart from these interactions, a recent
study on modifications to the pantothenoyl amide indicated that
interactions with this amide are also crucial for determining
(compared to the two-carbon linker in the -alanine moiety of
pantothenate), which may allow 9e to be accommodated in the
binding pocket without radically disturbing the interactions with
the carboxylate group. The importance of these interactions is
highlighted by the finding that simply converting the acid to a
methyl ester (as in 9b) was sufficient to decrease activity with
PaPanKIII below detection, indicating that the interactions
1
8
activity with SaPanKII. Interestingly, we show here that the
same SARs apply when a triazole group is used as an amide

A) EcPanKI
B) SaPanKII
C) PaPanKIII
Figure 3. Crystal structures of PanKs emphasizing ligand interactions observed in some crystallized complexes. A) EcPanK in complex
I
2
8
17
with pantothenate (PDB: 1SQ5); B) SaPanK in complex with phosphorylated N5-Pan (PDB: 4M7Y); C) PaPanKIII in complex with
II
2
9
pantothenate (PDB: 2F9W).
bioisostere (e.g. compounds 9f–k). The results show that
triazoles attached to the pantoyl amide with two carbon-linkers
study, as an example of a Gram-positive bacterium expressing a
type I PanK. No potent growth inhibitors were discovered,
suggesting that any inhibition of the PanKs or other CoA-
dependent targets were likely offset by the pantothenate present
in the rich media. Nonetheless, the results obtained with S.
aureus are interesting (Table S1). The most active compounds
were found to be triazoles 9h–j that all have a two carbon-linker
(9h–j) show high affinity for the enzyme (low K0.5 values), but
poor turnover (low kcat values). This is similar to what is observed
with the corresponding pantothenamides that were found to be
phosphorylated and then trapped in SaPanK ’s active site as the
II
1
7
phosphorylated products. In contrast, the triazoles attached to
the pantothenoyl amide via a one or three carbon-linker (9f,g,k)
show much better turnover, although at the cost of significantly
reduced affinity. This suggests that due to their comparatively
poor interactions with the enzyme, these compounds are
2
5
between the triazole and the pantoyl amide. These compounds
were designed to mimic N5-Pan and N4-Pan, two known
pantothenamides with antibacterial activity against S. aureus
9
,25,33
(Figure 4).
In comparison, the triazoles with a one carbon-
phosphorylated but then released. Docking results of compounds
linker (9f,g) or with a three carbon-linker (9k) show negligible
9
f, 9h and 9k (which only differ in the relative location of the
growth inhibition (Table S1). This finding is in agreement with
triazole) with SaPanK (PDB 4M7Y) are in agreement with our
II
enzymatic data. They suggest that all three compounds can be
accommodated in the binding pocket, with compound 9h
showing the lowest ∆G and a better alignment for hydrogen
bonds between the enzyme’s active site residues and the triazole
N-3 and CH, consistent with the triazole acting as a functional
mimic of the amide bond (Figure S1).
It is worth noting that although the N-substituent binding
pockets of EcPanK and SaPanK are both able to accommodate
I
II
a range of variations—an advantage for the design of
pantothenate antimetabolites—our results demonstrate that it is
possible to target either enzyme specifically. An example of such
targeted specificity is seen for compound 9c that is only a
substrate for SaPanK , while 9d is only acted on by EcPanK .
II
I
Finally, only PaPanKIII tolerated the removal of both geminal
methyl groups of pantothenate. Compound 9l was accepted as a
Figure 4. Two-carbon linker triazoles possibly mimicking the
pantothenamides N5-Pan and N4-Pan. Red highlights the
hydrogen bond acceptors, and blue highlights the hydrogen bond
substrate by PaPanK , although with some loss in affinity. This
III
is consistent with the binding pocket of PaPanK being tight and
III
a
donors. MICs shown are for S. aureus in rich medium.
more dependent on hydrogen bonding and ionic interactions than
hydrophobic ones, while the opposite trend is observed for
EcPanK and SaPanK . This result also implies that it may be
I
II
the analysis that the compounds showing antistaphylococcal
activity bind to enzyme in a manner that leads to the trapping of
the phosphorylated products on the enzyme (i.e. compounds
show high affinity and slow turnover)—as is the case for the
possible to design PaPanK -specific pantothenate
III
antimetabolites and/or inhibitors.
2
.4. Antimicrobial activity
1
7–18
antistaphylococcal N5-Pan.
Compounds 9a,e show better
The antibacterial activity of compounds 1a–l and 9a–l against
antistaphylococcal activity than 9b–d,l (Table S1). Since
E. coli (Gram-negative with a type I PanK), S. aureus (Gram-
positive with a type II PanK) and P. aeruginosa (Gram-negative
with a type III PanK) was next measured in rich media.
Enterococcus faecium was also included in the antibacterial
compounds 9a,e are better SaPanK substrates than 9b–d,l
II
(
Table 1), their superior antistaphylococcal activity might be
related to their better activity as SaPanK substrates, which
II

would likely translate into a more efficient formation of the
corresponding CoA antimetabolites. These compounds will cause
growth inhibition by interfering with downstream CoA-related
pathways.
Acknowledgments
We are grateful to Dr. Suzanne Jackowski for providing the
PaPanKIII expression plasmid, and Eric Ma for purifying
EcPanK . The China Scholarship Council is thanked for financial
support to J.G.
I
3
. Conclusion
Overall, a series of pantothenate analogues modified at
different positions were synthesized with convenient synthetic
methodology. Various substituents were introduced at the
primary hydroxyl group; which led to a molecule, 1g, able to
inhibit all three PanK types. Specific inhibitors were also
References
1
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I
for SaPanK . Our results suggest that when modifications are
introduced at the primary hydroxyl group of pantothenate,
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SaPanK might prefer negatively charged moieties. In contrast,
II
EcPanK has no preference between nitrogen or oxygen at this
I
position, while PaPanKIII prefers a nitrogen.
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Modifications at the secondary hydroxyl group of
pantothenate allow selectivity for EcPanK . Interestingly, when
I
the geminal dimethyl groups of pantothenate were replaced with
hydrogen atoms, the interaction of the resulting molecule (9l)
with both EcPanK and SaPanK were greatly affected, while the
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.
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effect was small for PaPanK , yielding a substrate that is
III
selective for this enzyme.
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Modifications at the -alanine moiety of pantothenate was a
more versatile strategy to differentiate activity and produced both
molecules that are specific for one type of PanK, as well as
compounds accepted as substrates by more than one PanK type.
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8–29
Consistent with the crystal structures,
the kinetic profiles of
9
1
1
.
de Villiers, M.; Barnard, L.; Koekemoer, L.; Snoep, J. L.; Strauss,
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these compounds imply that the pocket of PaPanKIII around the
terminal carboxylate of pantothenate is fairly tight and well-
defined, while that of EcPanK and SaPanK is more
I
II
0. Strauss, E.; Begley, T. P., The antibiotic activity of N-
pentylpantothenamide results from its conversion to ethyldethia-
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promiscuous, with the latter likely larger.
In recent years, research has revealed the important role of the
gut microbiome in health and disease. It is increasingly
recognized that broad-spectrum antibacterial agents lead to
severe imbalances in the normal gut microbiome, which is linked
3
4
to various diseases, and even to secondary infections by
3
5
Clostridium difficile (which harbors a type III PanK). The
3
6
1
2. Thomas, J.; Cronan, J. E., Antibacterial activity of N-
pentylpantothenamide is due to inhibition of coenzyme A
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6
–7
for the development of new antimicrobials. As a key enzyme
in the CoA biosynthetic pathway, PanK has attracted increasing
interest in recent years. Our use of pantothenate analogues as
probes to study the ligand preferences of PanK suggests possible
paths to design inhibitors or antimetabolites selective for an
organism with a particular type of PanK.
1
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1
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1
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Competing interests statement
The authors declare no competing interests.
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Funding sources
Staphylococcus aureus refractory to feedback regulation by
coenzyme A. Journal of Biological Chemistry 2005, 280 (5),
This work was financially supported by grants from the
Canadian Institute of Health Research (to K.A.) and from the
South African National Research Foundation (to E.S.).
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pantothenamide inhibitors of Staphylococcus aureus pantothenate
The following file is available free of charge. Supporting
Information.docx contains experimental protocol, compound
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characterization data and H NMR and C NMR spectra.

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