Antibiotic evaluation and in vivo analysis of alkynyl Coenzyme A antimetabolites in Escherichia coli

Article abstract of DOI:10.1016/j.bmcl.2008.07.078

Pantothenamides have been the subject of much study as potential inhibitors of CoA and carrier protein dependent biosynthetic pathways. Based on an initial observation of growth inhibition in Escherichia coli by 3, we have synthesized a small panel of pantetheine analogues and re-examined the inhibitory properties of this class of antibiotics with an emphasis on understanding the ability of these compounds to act as substrates of native CoA and carrier protein utilizing biosynthetic pathways. Our findings suggest that a secondary structure-activity relationship is an important factor in the antibiotic activity of these compounds.

Full text of DOI:10.1016/j.bmcl.2008.07.078

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5991–5994
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antibiotic evaluation and in vivo analysis of alkynyl Coenzyme
A antimetabolites in Escherichia coli
*
Andrew C. Mercer , Jordan L. Meier , Gene H. Hur, Andrew R. Smith, Michael D. Burkart
Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Pantothenamides have been the subject of much study as potential inhibitors of CoA and carrier protein
dependent biosynthetic pathways. Based on an initial observation of growth inhibition in Escherichia coli
by 3, we have synthesized a small panel of pantetheine analogues and re-examined the inhibitory prop-
erties of this class of antibiotics with an emphasis on understanding the ability of these compounds to act
as substrates of native CoA and carrier protein utilizing biosynthetic pathways. Our findings suggest that
a secondary structure–activity relationship is an important factor in the antibiotic activity of these
compounds.
Received 8 July 2008
Accepted 18 July 2008
Available online 24 July 2008
Keywords:
Fatty acid
Coenzyme A
Antibiotic
Ó 2008 Elsevier Ltd. All rights reserved.
Pantothenamides
Structure–activity relationships
Coenzyme A (CoA) is an essential cofactor in living systems, func-
tioning as the major acyl group carrier in numerous metabolic path-
ways.1 The bacterial CoA biosynthetic pathway was first elucidated
in Escherichia coli, where it was shown that five enzymes (CoaA,
CoaB, CoaC, CoaD, and CoaE) are responsible for the conversion of
pantothenate (vitamin B5) to CoA (Fig. 1A).2 While this pathway also
exists in humans, comparison of the individual enzymes of the pro-
karyotic and eukaryotic CoA biosynthetic pathways has shown that
the two demonstrate very little sequence homology.3 With reports
of bacterial resistance to traditional antibiotics becoming increas-
ingly common, the design of selective inhibitors of bacterial CoA bio-
synthesis presents a potentially novel antibiotic target.4
In addition to functioning directly as an acyl group carrier, CoA
is also the source of the 40-phosphopantetheine arm used for acyl
group transfer in fatty acid biosynthesis (Fig. 1A).5 In this pathway,
a phosphopantetheinyltransferase (PPTase) enzyme functions to
transfer the 40-phosphopantetheine arm of CoA to a conserved ser-
ine residue of an apo-acyl carrier protein (ACP). The free thiol of
this posttranslational modification is then used as the site of
acyl-intermediate tethering during the loading, condensation,
and reduction reactions necessary for production of fatty acids.
ACPs and peptidyl carrier proteins (PCPs) are used similarly in
polyketide and nonribosomal peptide biosynthesis.6
ification of ACPs with CoA analogues in vitro.7 When derivatized
with fluorescence or affinity tags, this property can be used for
the selective visualization and isolation of carrier protein utilizing
biosynthetic enzymes.8 Our group has had a long-standing interest
in using PPTase promiscuity as a strategy for the investigation of
primary and secondary biosynthetic pathways in bacterial organ-
isms, which has lead us to investigate methods for the manipula-
tion of intracellular CoA pool as a means of labeling carrier
proteins. As CoA analogues cannot cross the cell membrane due
to their strong negative charge, we have examined the utility of
CoA precursors as in vivo carrier protein labels.9
Perhaps the most thoroughly investigated CoA precursors to
date have been the antibacterial pantothenamides.10 This class of
antibiotics, typified by N5-Pan (1), has been shown to inhibit E. coli
and Staphylococcus aureus growth.11 Originally postulated as inhib-
itors of the pantothenate kinase (CoaA) enzyme, it has since been
shown that these compounds act as competitive substrates of
CoaA, and are believed to exert their antibacterial activity through
interference with fatty acid biosynthesis by labeling of the E. coli
fatty acid ACP (Fig. 1B).12,13
During our own studies of CoA precursors we encountered an
interesting phenomenon relevant to the continued development
and study of this antibiotic class. Through the synthesis and eval-
uation of a large number of CoA precursors (henceforth referred
to as pantetheine analogues), we identified a single compound
(2) capable of modifying the E. coli fatty acid ACP in the native
organism (Fig. 1B).14 Further, these studies showed that compound
2 was non-toxic to E. coli grown in minimal media at concentra-
tions >1 mM, while a structurally similar alkynyl pantetheine ana-
logue (3) possessed cytotoxic activity near equivalent to that of 1
In the past several years, it has been shown that many PPTases,
most notably Sfp from the surfactin synthetase pathway in B. sub-
tilis, have a relaxed substrate specificity which allows for the mod-
* Corresponding author. Tel.: +1 858 534 5673; fax: +1 858 822 2182.
E-mail address: mburkart@ucsd.edu (M.D. Burkart).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.078

5992
A. C. Mercer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5991–5994
Figure 1. (A) Pathway for CoA biosynthesis and posttranslational modification of carrier proteins. (B) Pantetheine analogues can act as alternate substrates for these
pathways to form CoA antimetabolites. These CoA analogues can be processed by PPTases to form crypto-carrier proteins. (C) Structures of pantothenate and pantetheine
analogues (1–15) examined in this study.
(Table 3). These observations ran contrary to expectations, as it
was anticipated that the most toxic pantetheine analogues would
be those which labeled the E. coli fatty acid ACP most efficiently,
according to the proposed mode of action of these compounds.13
These findings lead us to synthesize a small panel of pantethe-
ine analogues in order to examine in detail the influence of chain
length (4–9), oxidation state (10–11), and functionality (12–15)
on the ability to serve as CoA antimetabolites and inhibit growth
in E. coli (Fig. 1C).15 We first tested these compounds as substrates
of CoaA, the first enzyme of CoA biosynthetic pathway and the
rate-limiting step for the accumulation of CoA metabolites.16 In
past studies, we have correlated good turnover by CoaA with
in vivo CoA production and carrier protein modification.17 As can
be seen in Table 1, many of the analogues exhibit good turnover
with some (1–3, 5, 10–11, 13) approaching the kinetic efficiency
of pantothenate. When examining the effect of chain length on
turnover of alkynyl pantetheines, 4 shows very poor catalytic effi-
ciency due mainly to its high Km. This is indicative of weak binding,
a conclusion consistent with the crystal structure of E. coli CoaA
which shows the terminal acid of pantothenate having an impor-
tant role in binding. This can be replaced by interaction with an
amide which is missing in compound 4.4 In contrast, 3 and 5 show
catalytic efficiencies in the range of the native substrate, while 6–9
show a decrease in efficiency that correlates to the length of the
Table 1
Kinetic data for CoaA turnover of natural substrate (pantothenate) as well as pantetheine analogues 1–15a
Compound #
Km
(lM)
Vmax
(l
M sꢀ1
)
kcat (minꢀ1
)
kcat/Km (mMꢀ1 minꢀ1
)
Pantothenate
1
2
3
4
5
6
7
8
35.81 ( 6.00)
33.98 ( 7.11)
43.51 ( 3.4)
0.31 ( 0.01)
0.34 ( 0.02)
0.76 ( 0.03)
0.84 ( 0.05)
0.69 ( 0.01)
0.77 ( 0.04)
0.64 ( 0.12)
0.81 ( 0.07)
0.72 ( 0.07)
0.72 ( 0.07)
0.56 ( 0.05)
0.54 ( 0.04)
0.94 ( 0.04)
0.91 ( 0.05)
0.53 ( 0.03)
0.65 ( 0.03)
0.62 ( 0.02)
0.64 ( 0.04)
21.13 ( 1.93)
24.59 ( 2.88)
16.1 ( 0.53)
21.38 ( 2.11)
1.62 ( 0.61)
23.78 ( 4.23)
17.95 ( 3.41)
17.97 ( 3.75)
11.70 ( 2.22)
11.11 ( 1.53)
22.85 ( 1.96)
24.65 ( 2.88)
15.43 ( 1.52)
23.49 ( 2.33)
18.29 ( 1.20)
10.96 ( 1.49)
0.28 ( 0.002)
0.31 ( 0.02)
0.26 ( 0.05)
0.33 ( 0.03)
0.29 ( 0.03)
0.29 ( 0.03)
0.23 ( 0.02)
0.22 ( 0.02)
0.38 ( 0.02)
0.37 ( 0.02)
0.22 ( 0.01)
0.26 ( 0.01)
0.25 ( 0.01)
0.26 ( 0.02)
36.04 ( 6.05)
396.50 ( 123.7)
34.04 ( 10.72)
40.34 ( 12.95)
40.00 ( 14.26)
47.80 ( 14.62)
48.49 ( 10.99)
41.16 ( 5.94)
36.91 ( 7.52)
34.57 ( 6.07)
27.78 ( 5.21)
33.70 ( 3.98)
57.97 ( 12.01)
9
10
11
12
13
14
15
a
Values are means of three experiments, standard deviation is given in parentheses.

A. C. Mercer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5991–5994
5993
analogue. This decrease in efficiency is from both binding and turn-
over, as Km increases and Vmax decreases with the growing length
of the alkynyl pantetheine substrates. Compounds 8 and 9 high-
light another interesting trend seen in this panel, which is the det-
rimental effect of an amide bond at the natural thiol position of
pantetheine on CoaA turnover. This effect is most readily observed
by comparison of 12, 13, and 14, which demonstrates a steady de-
crease in catalytic efficiency with decreasing polarization of the
carbonyl (amide < carbamate < ester) at this position (see Table 1).
In comparing what effect degree of unsaturation has on turn-
over of pantetheine analogues by CoaA, the overall trend appears
to indicate marginally improved turnover for fully saturated al-
kyl-pantetheine analogues. While this effect is slight among those
pantetheine analogues terminating in propyl-derived chains (3, 10,
11), it can be clearly observed upon comparison of 1 to 6. A similar
effect is observed on comparison of amide bond-extended ana-
logues 12 and 15.
While kinetic values of pantetheine analogues with CoaA are a
good predictor of in vivo activity, many other factors, including cell
permeability and susceptibility to efflux pumps, impact the perfor-
mance of antimetabolites when administered to living cells. To
analyze the ability of these compounds to be processed by the
CoA biosynthetic pathway in vivo and to interact with carrier pro-
teins as CoA analogues, we utilized an in vivo assay.17 This assay
provides a qualitative measure of the ability of pantetheine ana-
logues to be processed by the endogenous CoA biosynthetic en-
zymes of E. coli by coupling CoA analogue production to the
modification of a carrier protein. To facilitate detection and isolate
CoA biosynthesis from variables such as carrier protein expression
and PPTase promiscuity, E. coli are first transformed with expres-
sion plasmids for a carrier protein, in this case the Fren-ACP from
the frenocylin polyketide synthase, as well as the PPTase Sfp, which
is known to have a very broad substrate specificity. After growth to
mid-log phase, the pantetheine analogue (1–15) is added at the
same time as IPTG, which induces expression of the reporter sys-
tem.15 Compounds that exhibit uptake and processing by the na-
tive E. coli CoA biosynthetic pathway produce modified ACPs,
which demonstrate a mass shift characteristic of posttranslational
modification by each analogue, and can be observed by MALDI-TOF
(Table 2).
have on antibiotic activity, we determined the MIC values for 1–
15 using E. coli K12 grown in both minimal (M9) media, as well
as in a richer, 1% tryptone broth which had been used to determine
MIC values in an earlier study of pantothenamides.11,13
Inspecting the results, all of the analogues tested showed greater
growth inhibition in minimal media compared to rich media (Table
3). These resultsshow a direct correlationbetweentoxicity andCoaA
kinetic profile for these compounds. This is to be expected, as it has
previously been shown that CoaA is the rate-limiting step for CoA
biosynthesis in vivo, and the antibacterial activity of these com-
pounds is believed to be dependent on their in vivo transformation
to CoA analogues. The major outliers in this respect are 2 and 13,
which possess good kinetics but do not show inhibition of E. coli at
concentrations up to 500 lM even in minimal media. Additional evi-
dence that these compounds act as CoA antimetabolites was pro-
vided by the observation that the inhibitory effects of the most
toxic members of this panel (1, 3, 5–6, 10–11) were greatly de-
creased by addition of the CoA precursors pantothenate and b-ala-
nine to the growth medium (Table 3). Among the alkynyl
analogues which initially inspired this study (3, 5–9), an increasing
MIC value is observed with growing chain length, mirroring the de-
clinein catalyticefficiencyobservedamongthisgroup. Interestingly,
among alkyl pantetheine analogues of the same chain-length (3 and
11, 1 and 6), changing the oxidation state from an alkyne to a satu-
rated alkyl chain lowers the MIC by a factor of two to four. However,
while 11 is sixfold more active than 1 in minimal media, administra-
tion of these same compounds to E. coli grown on rich media shows
11 to be at least 10ꢁ less toxic under these conditions.
The kinetic profiles, in vivo analysis, and inhibitory data gener-
ated here all support the previously held hypothesis that the antibi-
otic activity of pantetheine analogues is due to the production of CoA
analogues in vivo.12 However, the finding that saturated and unsat-
urated pantetheine analogues demonstrate rates of CoaA turnover
within error of one another (i.e., 3 and 11), yet show drastically dif-
ferent MICs suggests that CoA analogue production alone is not suf-
ficient for antibacterial activity. Based on our results it appears that
of the pantetheine analogues processed efficiently by CoaA, those
terminating in fully saturated alkyl groups are ideal for activity,
while substitution by unsaturated alkynyl chains and polar terminal
groups on the pantetheine chain (i.e., 2 and 13) results in decreased
orno growthinhibition. Thissuggestsasecondarystructure–activity
relationship for pantetheine analogue inhibition, in which one set of
structural characteristics is necessary for biosynthetic processing
and formation of CoA or ACP-analogues in vivo, while the identity
Having confirmed that the majority of these compounds are
capable of formation of CoA analogues in vivo, we sought to corre-
late our findings with their antibacterial activity in native E. coli. To
investigate the effects that additives present in the media might
Table 3
Table 2
Minimum inhibitory concentrations of pantetheine analogues 1–15 to E. coli grown in
MALDI-TOF data for in vivo modification of Fren-ACP by compounds 1–15
different media and effect of additives
Compound #
apo
Modified
Difference
Expected
Media compound #
M9
Tryptone
M9 + Pana
M9 + b-Alab
MIC (
lM)
MIC (
lM)
MIC (
lM)
MIC (lM)
None (control)
1
2
3
4
5
6
7
8
8663
8660
8663
8663.7
8661.1
8664.9
8662.2
8665.8
8663.4
8666
8658
8660
8660
8660
8660
8666
9000a
9011
9018
8982
—
8996
9008
337a
351
355
318.3
—
331.1
345.8
360.1
401.6
376b
321
342a
351
358
319
248
333
347
361
404
376
321
323
380
381
396
376
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
6
>500
4
500
7.5
15
500
>500
>500
3
50
500
ndc
>500
nd
>500
>500
nd
nd
nd
250
500
nd
250
nd
>500
nd
>500
>500
nd
nd
nd
125
250
nd
>500
>500
>500
>500
>500
>500
>500
>500
>500
500
9025.9
9065
9
9042 & 9350b
8979
8982
9042
10
11
12
13
14
15
322
382
386
398
1
>500
500
>500
500
>500
>500
>500
>500
9046
9058
9039
nd
nd
nd
nd
nd
nd
373
a
a
Modification by native CoA of E. coli results in the expected mass shift.
Compound 9 gives a large peak at 9350 in addition to the expected peak at
Pan, addition of 1 mM pantothenate to growth medium.
b-Ala, addition of 1 mM b-alanine to growth medium.
nd, not determined.
b
b
c
9042, possibly due to matrix interactions with the unsaturated activated alkyne.

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A. C. Mercer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5991–5994
of the terminal group facilitates interaction of the CoA or ACP-ana-
logue with a biologically relevant target.
References and notes
1. Leonardi, R.; Zhang, Y. M.; Rock, C. O.; Jackowski, S. Prog. Lipid Res. 2005, 44,
125.
2. Strauss, E.; Kinsland, C.; Ge, Y.; McLafferty, F. W.; Begley, T. P. J. Biol. Chem.
2001, 276, 13513.
In E. coli, fatty acid ACP is estimated to be present in the cytosol
at concentrations approaching 1 mM. This abundance may explain
the observation that 2 is capable of ACP modification in native E.
coli without toxicity. Why then do alkyl pantetheine analogues 1
and 10–11 show increased inhibitory properties? Based on their
kinetics with CoaA, these analogues seem unlikely to modify ACP
at substantially higher levels than 2 in vivo. More likely, and in
agreement with the secondary structural features of antimicrobial
pantetheine analogues observed here, is the hypothesis that the
activity of alkyl pantetheine analogues is due to the differential
activity of alkyl-ACP-prodrugs to bind and disrupt the associated
lower abundance enzymes of fatty acid biosynthesis. Further eluci-
dation of this process may have important implications for design
of new members of this antibiotic class.
3. Gerdes, S. Y.; Scholle, M. D.; D’Souza, M.; Bernal, A.; Baev, M. V.; Farrell, M.;
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Acknowledgment
This work was funded by NIH RO1GM075797; JM was sup-
ported by T32DK007233.
14. Mercer, A. C.; Meier, J. L.; Torpey, J. W.; Burkart, M. D. Submitted for
publication.
15. See Supporting information for synthetic procedures and data as well as assay
details.
Supplementary data
16. Jackowski, S.; Rock, C. O. J. Bacteriol. 1981, 148, 926.
17. Meier, J. L.; Mercer, A. C.; Rivera, H., Jr.; Burkart, M. D. J. Am. Chem. Soc. 2006,
128, 12174.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.07.078.