New insights into the bacterial RNA polymerase inhibitor CBR703 as a starting point for optimization as an anti-infective agent

Article abstract of DOI:10.1128/AAC.02600-14

CBR703 was reported to inhibit bacterial RNA polymerase (RNAP) and biofilm formation, considering it to be a good candidate for further optimization. While synthesized derivatives of CBR703 did not result in more-active RNAP inhibitors, we observed promising antibacterial activities. These again correlated with a significant cytotoxicity toward mammalian cells. Furthermore, we suspect the promising effects on biofilm formation to be artifacts. Consequently, this class of compounds can be considered unattractive as antibacterial agents. Copyright

Full text of DOI:10.1128/AAC.02600-14

AAC Accepts, published online ahead of print on 12 May 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.02600-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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New insights into the bacterial RNA polymerase inhibitor CBR703
as a starting point for optimization as an anti-infective agent
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Weixing Zhu,1 Jörg Haupenthal,1 Matthias Groh,1 Michelle Fountain3 and Rolf W.
Hartmann1, 2*
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1 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department of Drug Design
and Optimization and 2 Pharmaceutical and Medicinal Chemistry, Saarland University, Campus
C2.3, D-66123 Saarbrücken, Germany. 3 Helmholtz Centre for Infection Research,
Inhoffenstraße 7, 38124 Braunschweig, Germany.
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* Corresponding author. Fax: (+49) 681 302 70308 E-mail: rolf.hartmann@helmholtz-hzi.de
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ABSTRACT
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CBR703 was reported to inhibit bacterial RNA polymerase (RNAP) and biofilm formation,
considering it to be a good candidate for further optimization. While synthesized derivatives of
CBR703 did not result in more active RNAP inhibitors, we observed promising antibacterial
activities. These again correlated with a significant cytotoxicity towards mammalian cells.
Furthermore, we suspect the promising effects on biofilm formation to be artifacts. Consequently,
this class of compounds can be considered unattractive as antibacterial agents.
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Bacterial RNA polymerase (RNAP) is essential for bacterial growth and survival and thus an
attractive target for drug development (1, 2). Along with the recently FDA approved fidaxomycin
(3), the rifamycins, applied as first line antituberculosis drugs, are the only RNAP inhibitors that
are in clinical use (2). However, similar to other anti-infectives, the use of rifamycins resulted in
the occurrence of resistant bacterial strains (1, 4 – 7), which represents a remarkable threat to
public health (8, 9). Consequently, there is need to focus on novel promising inhibitors. Recently,
interesting peptidic and peptidomimetic (10 – 12) as well as non peptidic small molecule RNAP
inhibitors (13 – 18) have been described. Another example is CBR703 (Fig. 1), whose
mechanism of action is reported to be different from that of the rifamycins (19, 20). This
compound has been identified in a high throughput screening searching for small molecule
inhibitors of RNAP (19). Two more potent analogs in this report reveal the potential of
optimizing CBR703 by structural enlargement. Furthermore, pursuing the hypothesis that RNAP
is of particular importance for bacterial survival in biofilms, Villain-Guillot et al. showed
CBR703 to significantly reduce the Staphylococcus epidermidis biofilm mass (21). We therefore
considered CBR703 to be a promising starting point for drug development. Consequently, we
focused on CBR703 to perform systematic modifications on its core structure, aiming to obtain a
more appropriate starting point for further structural optimization.
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Detailed information concerning the materials and methods used in synthesis and biology can
be found in the Supplemental Material.
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In total, 30 final compounds and 24 intermediates were obtained and tested for E. coli RNAP
inhibition and their ability to inhibit the growth of E. coli TolC (Supplemental Material Table S1
– 3). According to their structures, the synthesized derivatives can be divided into three groups
with modifications in A, B or C (Fig. 1). 1 – 25 (Scheme S1) with introduction of substituents
into the aromatic moieties (A or B) were prepared by condensation of an intermediate amide with
hydroxylamine (22, 23). In order to assure an appropriate coverage of lipophilic and electronical
properties, the substituents were chosen rationally from all quadrants of a Craig Plot (e.g.
Hansch-Fujita π versus σ constant) (24). The results (Table S1) showed that compounds 1 – 25
display a decreased RNAP inhibition compared to CBR703 with the exception of two
compounds (18 and 19) with similar activity (IC50s in the range of 20 µM). As reported (19),
there were two more potent CBR703 analogs with larger size, one of which was optimized by
replacing the linker amidoxime with a pyrazole system. To investigate this structural
modification, 26 – 30 with a different linking part (C) have been synthesized (Table S2).
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Remarkably, in our case, replacement of the amidoxime moiety by other functional groups
including N-heterocycles led to a decrease or complete loss of activity. Additionally, all amide
intermediates turned out to be inactive against RNAP (Table S3). Surprisingly, 11 compounds
including intermediates with little or even no RNAP inhibition showed a stronger antibacterial
potency in E.coli TolC than CBR703. 3a with a MIC of 2 µg/mL was even more potent than
rifampicin. The fact that no correlation between RNAP inhibition and antibacterial activity (Table
S1 – 3) could be observed led us to the conclusion that additional mechanisms besides RNAP
inhibition must be responsible for the antibacterial activity.
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To obtain further information about the antibacterial profiles, four compounds (Fig. 1) were
selected based on the results of the previous experiments (Table S1 – 3), and compared with
reference compounds. In a first step, the effects of these compounds on the growth of E. coli K12,
Pseudomonas aeruginosa PAO1 (PAO1), Bacillus subtilis (B. subtilis) and Staphylococcus
aureus (S. aureus) were investigated (Table 1). Notably, 7 (best compound against E.coli TolC
bearing an amidoxime group) and 19 (most active RNAP inhibitor) only showed moderate
activity against B. subtilis. 3a (most active against E.coli TolC) exhibited rather potent activities
against B. subtilis and S. aureus. For 26 (the only compound with RNAP inhibition after
replacement of the amidoxime linker), we observed no detectable activities against Gram-
positives. None of the compounds inhibited the growth of the Gram-negative strains K12 and
PAO1. In addition, the toxicity of the inhibitors towards mammalian cells was tested using the
Human Embryonic Kidney (HEK) 293 cell line. Interestingly, the most active compound 3a in
the MIC experiment showed a significant cytotoxicity and also the other tested compounds were
at least moderately toxic (Table 2). As it is known that lipophilic compounds bind to serum
proteins, which are also present in our MTT assay as a component of fetal calf serum (FCS), we
added the same amount of FCS (10 %) to the bacterial growth medium and performed the MIC
determinations in E.coli TolC. Surprisingly, the antibacterial activity of the tested compounds
was abolished or drastically reduced (Table S4). This finding led to the assumption that the
cytotoxicities of our compounds are even more pronounced in the absence of serum.
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As it had been shown that CBR703 efficiently eradicated biofilm-embedded bacteria (21), we
considered that this effect could be due to Fe(III) chelation (25, 26). The fact that the amidoxime
moiety plays a prominent role for the activity in our compounds and the well-known property of
amidoxime functional groups to complex Fe(III) gave rise to the presumption that the
amidoximes display their antibacterial effect due to such a complexation (27, 28). Consequently,
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we examined this hypothesis. Firstly, the ability of CBR703 to form Fe(III) complexes was
confirmed by a color change reaction (29). After addition of a CBR703 solution, the brown red
FeCl3 solution turned to blue while this change was not observed after addition of 26 (Table S5).
In a following step, the complex stability constants were determined by potentiometric titration.
Thereby it was uncovered that only under acidic conditions (pH = 4) formation of Fe(OH)3 was
observed. This means that under physiological conditions CBR703 cannot form stable Fe(III)
complexes. These results were supported by biological tests which were performed in parallel.
Indeed, addition of Fe(III) had an effect on the anti-TolC-activity of the positive control
deferoxamine mesylate (DFO) - a known iron chelator with antibacterial activity (30) - but not on
CBR703, leading to our conclusion that the antibacterial effects of CBR703 are not attributed to
iron complexation (Fig. S1). Interestingly, each of the three most antibacterial compounds (3a,
10a, 21a) possesses two strong electron withdrawing- (leads to polarity decrease) and highly
lipophilic CF3 groups which might be the reason for their antibacterial potency. Such properties
could facilitate cell penetration and furthermore result in non-specific inhibition of a variety of
other enzymes.
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During the determination of MIC values we found that CBR703 showed a slight precipitation
at 100 µg/mL while in the literature its MIC was determined to be 100 µg/mL (21). Beyond that a
significant effect on Staphylococcus epidermidis biofilm was reported at concentrations between
100 and 400 µg/mL. At these concentrations we observed a strong and concentration-dependent
precipitation of CBR703 and selected derivatives in Mueller Hinton Broth (MHB) (Fig. 2A and
Fig. S2), the medium used in literature (21). Nevertheless, we evaluated all compounds on S.
aureus biofilms with concentrations in a soluble range, but without observing an effect. At higher
concentrations (100 – 400 µg/mL) CBR703 and its derivatives (e.g. 7 and 19) showed a clear
reduction in biofilm formation (Fig. 2B), indicating a correlation between anti-biofilm activity
and precipitation.
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In this work we designed and synthesized derivatives of CBR703 as follow up work to a
published paper (19) aiming to optimize their promising biological effects by modifying the core
structure. However, no compound showed an enhanced RNAP inhibition. Nevertheless, in some
cases we observed promising antibacterial activities. These again turned out to correlate with a
significant cytotoxicity towards HEK 293 cells. Furthermore, the reported effects on biofilm
formation, which were one of the main reasons for choosing CBR703 as a starting point, were
suspected to be artifacts due to compound precipitation. This finding should be a reminder to the
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scientific community to be cautious with published data as they could be artifacts (31).
Consequently, we rank this class of compounds as unattractive for the development as
antibacterial agents.
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ACKNOWLEDGMENTS
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W. Zhu thanks China Scholarship Council for her PhD fellowship. The authors thank Jeannine
Jung and Jannine Ludwig for technical assistance, Professor Dr. Kaspar and Dr. Bernd
Morgenstern from the Department of Chemistry (Saarland University, Germany) for the
determination of complex stability constants, Dr. Werner Tegge from Helmholtz Centre for
Infection Research (Braunschweig, Germany) for supporting M. Fountain’s biofilm tests and
Professor Witte from the Robert Koch Institute (Wernigerode, Germany) for kindly providing the
MSSA ST30 strain.
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Fig. 1 CBR703 and the most potent compounds in different classes: 7, best compound against
E.coli TolC bearing an amidoxime group; 19, most RNAP inhibitory derivative; 26, the only
RNAP inhibitor after replacement of the amidoxime linker; 3a, most active against E. coli TolC.
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Fig. 2 Correlation between precipitation and biofilm mass. (A) Concentration dependent
precipitation of CBR703, 7 and 19 in MHB. (B) Quantification of the washed biofilm mass. A
complete biofilm reduction can only be observed at concentrations at which precipitates have
formed. The most prominent effect can be observed for 7.
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TABLE 1 RNAP inhibition and antibacterial profile of selected compounds
MIC (µg/mL)a
% Inhibition
of E. coli
RNAP
compound
Gram negative
Gram positive
E. coli TolC E. coli K12 PAO1
B. subtilis
S. aureus
(at 50 M)
18 Mb
35
CBR703
14
9
>25
>25
>50
>25
>25
7
>25
>25
>50
>25
>25
13
>25
23
43
>25
5
>25
>25
>50
>25
11
7
19
26
19 Mb
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21
24
2
3a
n.i
Rifampicin
24 nMb
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0.02
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No correlation between RNAP inhibition and antibacterial activities was observed, suggesting
that the antibacterial activity was due to a mechanism other than RNAP inhibition. The SD in
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these experiments was < 25 % (most cases: < 15 %). >: MIC-determination was limited due to
insufficient solubility of the compound; b: IC50 value.
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TABLE 2 Investigation of cytotoxicity in HEK 293 cells.
compound
LD50 24 h (µM)
LD50 72 h (µM)
CBR703
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43
34
25
15
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40
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38
13
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0.3
7
19
26
3a
Rifampicin
Doxorubicin
>100
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The most potent antibacterial compound 3a was also the most toxic one. Rifampicin: negative
control; Doxorubicin: positive control; LD: lethal dose.
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A
OD600
CBR703
7
19
0.8
0.6
0.4
0.2
0
25 50 75 100 150 200 300 400 Conc. [µg/mL]
B
CBR-703
7
19
0
0.7 1.6 3.1 6.3 12.5 25 50 100 200 400 0 Conc. [µg/mL]
Fluorescence 485 / 517 nm
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