Ribosome-Templated Azide-Alkyne Cycloadditions Using Resistant Bacteria as Reaction Vessels: In Cellulo Click Chemistry

Article abstract of DOI:10.1021/acsmedchemlett.8b00248

In situ click chemistry has been a powerful method for fragment-based drug design since its discovery in 2002. Recently, we demonstrated that the bacterial ribosome can template the azide-alkyne cycloaddition reaction to expedite the discovery of novel antibiotics. We now report this process can be performed in an antibiotic-resistant bacterial cell. The corresponding triazole products formed in cellulo are potent antibiotics that inhibit bacterial growth; moreover, the potency of each cycloadduct can be visualized using the traditional MIC assay in a 96-well plate format. We characterized the in cellulo clicked products by independent chemical synthesis and LC-MS analysis, which showed that mass count percent increase was directly proportional to 1/MIC. In other words, potent compounds detected by MIC were formed in greater amounts. Control experiments unambiguously showed the ribosome was responsible for templating triazole formation. Significantly, our method (1) obviates the need to isolate bacterial ribosomes; (2) could be applied to different bacterial strains, which broadens the scope and facilitates the discovery of narrow-spectrum antibiotics; and (3) does not require the knowledge of mode-of-action and thus could uncover novel antibiotic targets. We believe this method could be expanded and implemented as a novel approach for antibiotic drug discovery.

Full text of DOI:10.1021/acsmedchemlett.8b00248

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Letter
Ribosome-templated azide–alkyne cycloadditions using
resistant bacteria as reaction vessels: in cellulo click chemistry
Xiao Jin, Samer S. Daher, Miseon Lee, Bettina A. Buttaro, and Rodrigo B. Andrade
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00248 • Publication Date (Web): 13 Aug 2018
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Ribosome-templated azide−alkyne cycloadditions using resistant
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Xiao Jin,1 Samer S. Daher,1 Miseon Lee,1 Bettina Buttaro,2 and Rodrigo B. Andrade1,*
1 Department of Chemistry, Temple University, Philadelphia, PA 19122
2 Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania
19140
In situ click chemistry, in cellulo click chemistry, macrolide antibiotic, ribosome, solithromycin
ABSTRACT: In situ click chemistry has been a powerful method for fragment-based drug design since its discov-
ery in 2002. Recently, we demonstrated that the bacterial ribosome can template the azide−alkyne cycloaddi-
tion reaction to expedite the discovery of novel antibiotics. We now report this process can be performed in an
antibiotic-resistant bacterial cell. The corresponding triazole products formed in cellulo are potent antibiotics
that inhibit bacterial growth; moreover, the potency of each cycloadduct can be visualized using the traditional
MIC assay in a 96-well plate format. We characterized the in cellulo clicked products by independent chemical
synthesis and LC−MS analysis, which showed that mass count percent increase was directly proportional to
1/MIC. In other words, potent compounds detected by MIC were formed in greater amounts. Control experi-
ments unambiguously showed the ribosome was responsible for templating triazole formation. Significantly,
our method (1) obviates the need to isolate bacterial ribosomes; (2) could be applied to different bacterial
strains, which broadens the scope and facilitates the discovery of narrow-spectrum antibiotics; and, (3) does
not require the knowledge of mode-of-action and thus could uncover novel antibiotic targets. We believe this
method could be expanded and implemented as a novel approach for antibiotic drug discovery
.
The alarming threat of bacterial resistance over the last
few decades has raised significant concerns among those
working in the public health sector. The Centers for Dis-
ease Control and Prevention reported a rapid increase in
the rate of mortality associated with bacteria resistant to
antibiotic treatment. Of the two million people diagnosed
with severe resistant bacterial infections in the United
States every year, approximately 23,000 die as a result of
ineffective antibiotic therapy. Since bacteria are naturally
subject to spontaneous whole genome mutagenesis and
can easily transfer antibiotic resistant genes to other bac-
teria by means of horizontal gene transfer, antibiotic re-
sistance is unavoidable.1,2 New antibiotics—and strategies
for their efficient discovery and procurement—are des-
perately needed to tackle this issue.3-5
Building on pioneering efforts by Sharpless,8 in situ click
chemistry has been successfully applied to many other
targets including carbonic anhydrase,9 HIV protease,10 chi-
tinase,11,12 histone deacetylase,13 and DNA.14 In 2015,
Heath employed in situ click chemistry to discover in-cell
inhibitors of botulinum neurotoxin15 and found a selective
inhibitor of a single point mutation of the Akt1 epitope.16
In 2016, we demonstrated that in situ click chemistry
can be used in the discovery of novel macrolide antibiotics
by employing the target—the bacterial ribosome—as a
catalyst in the presence of azide- and alkyne-functionalized
building blocks. As proof of concept, we showed that best-
in-class ketolide solithromycin (3) could be prepared in
situ from azide 1 and 3-ethynylaniline (2) (Figure 1A).17
To date, this is the largest and most complex target to be
used to template the Huisgen cycloaddition. Therein, it was
shown that the ribosome recognized and directed the pre-
cursor subunits in the proper orientation to generate
“clicked” triazole products selectively. The clicked prod-
ucts are macrolide inhibitors that target 50S ribosomal
subunit. For the in situ click method, we correlated the
extent of triazole formation with ribosome affinity as re-
vealed by measured Kd values. By comparing the mass
count percent increase of each compound in a multicom-
ponent in situ click assay, we can screen those compounds
rapidly and efficiently.
The advent of click chemistry has made a profound im-
pact on fragment-based drug discovery.6 In 2002, Sharp-
less and co-workers made a significant contribution when
they established the first in situ click method wherein the
Huisgen [3+2] cycloaddition between azide- and alkyne-
functionalized fragments was templated by the enzyme
acetylcholinesterase.7 In this manner, the two fragments
that bind in proximity are joined not by Cu(I) but by the
biological target (e.g., enzyme), which pays the entropic
penalty of bringing the azide and alkyne together to form
the triazole product.
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Figure 1. Ribosome-guided azide−alkyne (A) in situ and (B) in
cellulo click chemistry platforms for drug discovery.
well-established MIC assay. The MIC is the lowest concen-
tration of a compound that inhibits bacterial growth.
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The strain we identified for proof-of-concept studies was
the resistant Gram-positive bacterium Staphylococcus au-
reus UCN18, which carries a point mutation at position
2059 (i.e., A2059G). X-ray crystallographic studies of the
bacterial ribosome showed that mutation at this specific
location conferred resistance towards macrolides.22 Specif-
ically, ketolides possessing aromatic side-chains such as
telithromycin were able to overcome A2059G resistance
by making additional contacts within the macrolide bind-
ing site. We reasoned that this difference in potency could
be leveraged by using in cellulo click chemistry (ICCC)
wherein the ribosomes in the resistant bacteria template
the azide−alkyne cycloaddition and make their own inhibi-
tor. Indeed, the MIC of azide-functionalized macrolide 1
against S. aureus UCN18 was 256 ꢀg/mL (in the lag phase)
whereas the MIC of solithromycin (3), derived from the
click reaction of 1 and 3-ethynylaniline (2), was 2 ꢀg/mL.
In addition, the MIC of 3-ethynylaniline (2) was tested and
found to be 21.5 mg/mL. The MIC experiments with the
individual azide and alkyne fragments also confirmed an
important requirement for ICCC; namely, both fragments
must be able to enter the bacterial cell by active transport
or passive diffusion and engage the ribosome.
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In 2014, Disney and co-workers took the in situ click one
step further by using the whole cell as a reaction vessel
and expressing pathogenic RNA loops to catalyze the syn-
thesis of toxic RNA inhibitors (i.e., in cellulo click chemis-
try) from azide- and alkyne-functionalized fragments.18
Significantly, this publication verified that the in situ click
approach could be viable in cellulo. A major motivation for
Disney’s development of in cellulo click was the potential
solution to a potency versus permeability tradeoff. That is,
low molecular weight molecules with weak target affinity
are more cell permeable than those with high molecular
weight that have stronger target affinity. By having the
target inside the cell carry out the in situ click reaction in
cellulo, the high molecular weight permeability problem is
solved.
Preliminary ICCC experiments were done in a binary
fashion utilizing azide-functionalized macrolide 1 and 3-
ethynylaniline (2) in the presence or absence of bacteria.
We hypothesized that wells containing bacteria would
template the in situ synthesis of solithromycin and result
in a lower MIC value whereas those wells lacking the bac-
teria would not. To test this hypothesis, we carried out in
cellulo click experiments based on the principle of a MIC
assay using 96 well plates (Figure 2). First, a fixed sub-
lethal concentration of azide 1 [128 ꢀg/mL] was added to
rows B through E whereas only BHI media was added to
rows A and F as controls. Second, a stock solution of alkyne
(21.5 mM in DMSO) was added to rows A, C, D, and E by
two-fold serial dilution beginning with column 8. Third, an
overnight culture of S. aureus UCN18 was diluted (1:1000
in BHI media) and added to all rows except row D, which
was used to quantify the background Huisgen reaction.
Most recently, Sellstedt elegantly demonstrated that bo-
vine carbonic anhydrase II (bCAII) found in red blood cells
could be inhibited in cellulo.19 Significantly, the use of mul-
ti reaction monitoring (MRM) mass spectrometry analysis
and deuterium-labeled internal standards were key to re-
capitulating the in situ click formation of a powerful bCAII
inhibitor first reported by Kolb.20
Our motivations for developing in cellulo click chemistry
(ICCC) were multifold. First, the method obviates the need
to isolate bacterial ribosomes, which can be tedious and
requires specialized equipment and expertise. Second, the
method could be applied to different bacterial strains,
which broadens scope and facilitates the discovery of nar-
row-spectrum antibiotics.21 Third, the method could be
used to selectively target resistant bacterial strains over
the wild-type and accordingly expedite the drug discovery
process. Based on our in situ click method with bacterial
ribosomes, we proposed that by employing macrolide-
resistant bacteria as reaction vessels, we would leverage
the ribosome as a catalyst to synthesize novel antibiotics
by combining azide and alkyne fragments without the need
to isolate the ribosomes themselves. Once formed in cellu-
lo, the antibiotic would inhibit bacterial growth. In addi-
tion, the potency of the triazole products could be visually
evaluated directly from a 48- or 96-well plate using the
Figure 2. Format of in cellulo click experiments based on the
MIC assay using 48- or 96-well plates.
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Scheme 1. (A) Overview of in cellulo click experiments. (B) Structures of alkyne fragments in the library and triazoles de-
rived from in situ click experiments with MIC values of 1,4-triazoles 3, 14−23 in square brackets.
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The rationale for dilution was so that bacteria would en-
ter the lag phase wherein the highly active ribosomes of a
manageable population of bacterial cells would catalyze
the click reaction in cellulo. We reasoned that during the
exponential phase, bacteria would be dividing at a much
higher rate than the competing click reaction, thus compli-
cating the detection of bacterial inhibition. If a potent anti-
biotic was formed from the click reaction during the lag
phase, it would inhibit ribosomal activity and lead to bac-
terial growth inhibition, which could be detected by the
naked eye. Moreover, the triazole (i.e., clicked) product
could be detected and quantified by LC−MS.
In addition, mass counts (i.e., area) corresponding to
solithromycin in these experiments were similar to those
obtained from the background Huisgen cycloaddition (row
D).
Figure 3. In cellulo click and control experiments with 3-
ethynylaniline.a-c
The results of preliminary experiments are shown in
Figure 2 (see SI for details). While 3-ethynylaniline (2) has
a high MIC, bacterial inhibition was observed at 21.5 mM
in well A8. As expected, no bacterial inhibition was ob-
served in row B where a sub-lethal concentration of azide
1 was employed. The in cellulo click experiment conducted
in row C (i.e., the combination of bacteria, azide, and a con-
centration gradient of alkyne from lowest in column 1 to
highest in column 8) showed that bacterial inhibition oc-
curred in well C6, which was consistent with our hypothe-
sis that the ribosomes are templating the Huisgen cycload-
***
***
C6
D6
E6
a
b
Azide/alkyne/bacteria in well C6; Azide/alkyne without
c
bacteria in well D6; Azide/alkyne/bacteria/AZY in well E6.
*** indicates p < 0.001 as per two-tailed Student’s t-test.
dition in cellulo
.
To verify that cycloadduct formation by in cellulo click
chemistry was responsible for the observed MIC difference
and not a synergistic effect of the individual fragments, we
performed LC−MS analysis (Figure 3).23 Specifically, we
identified solithromycin (3) in well C6 by both retention
time and mass-to-charge ratio (m/z) when compared to an
authentic sample prepared by Cu(I)-catalysis.24 To confirm
that solithromycin (3) formation was catalyzed by the ri-
bosome in cellulo as opposed to another bacterial target or
a component in the BHI media (row E), we performed the
in cellulo click experiment in the presence of ribosomal
inhibitor, azithromycin (7.04 mM, see SI for details). By
saturating the ribosomes with azithromycin (AZY), binding
to azide 1 and the attendant in cellulo click reaction are
precluded. We recently employed azithromycin in negative
control experiments in the development of the ribosome-
templated in situ click method.17
After establishing the viability of in cellulo click chemis-
try (ICCC) for the discovery of novel antibiotics to treat
resistant bacterial strains, we selected a library of twelve
alkynes to employ our method (Scheme 1). The library
featured ten aromatic alkynes, one of which was heteroar-
omatic (i.e., pyridine 8), in addition to piperazine 13 and
aliphatic alcohol 10. The experimental protocol followed
the 96-well format outlined in Figure 2, and each experi-
ment was run six times for reproducibility (see SI for de-
tails). The results are shown in Figure 4. The presence of
triazole cycloadducts 3 and 14−23 formed in cellulo was
confirmed with LC−MS by comparison with authentic sam-
ples prepared independently by Cu(I) catalysis, which
were also used to determine antibacterial activity against
S. aureus UCN18 in MIC assays (Scheme 1B).25
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Clicked triazole products displaying greater potency (i.e.,
and in cellulo click chemistry that possess greater target
affinity result in the formation more triazole product.
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antibacterial activity due to ribosome inhibition) will pos-
sess lower MIC values. A central premise of in situ click
chemistry is that targets (i.e., catalysts) favor the for-
mation of more potent inhibitors, which is registered by
mass spectrometry as higher mass count (Figure S1).6-8
Indeed, of the eleven triazoles prepared by ICCC with S.
aureus UCN18, solithromycin (3) with an MIC of 2 ꢀg/mL
was formed in the greatest amount. In decreasing amounts,
triazoles 21 (4 ꢀg/mL) was formed next, followed by tria-
zole 19 (8 ꢀg/mL) then 14 (8 ꢀg/mL).
Beyond the aforementioned advantages of in cellulo ver-
sus in situ click, the isolation of targets from pathogenic
strains (for antibacterial drug discovery) is avoided. Fur-
thermore, knowledge of the drug target (i.e., mechanism of
action) is not required since any enzyme, in principle, can
catalyze triazole formation from the corresponding azide-
and alkyne-functionalized fragments. Accordingly, this
agnostic approach can be applied to discover new antibiot-
ics with established targets (e.g. ribosome, cell wall, topoi-
somerase) or more significantly, uncover novel antibiotic
targets.
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Figure 4. Results of ICCC experiments showing mass
count percent increases of triazoles 3, 14−23 for the library
of eleven alkynes 2, 4−13.
Figure 5. Correlation between MIC and mass count per-
cent increase.
y = 0.0021x − 0.014
R2=0.96
3
14 15 16 17 18 19 20 21 22 23
Triazoles formed by ICCC
In summary, we have demonstrated that the ribosome-
guided in situ Huisgen [3+2] cycloaddition (click) chemis-
try can be performed in cellulo wherein bacterial cells
serve as reaction vessels. Proof-of-concept studies were
performed with the resistant Gram-positive bacterium
Staphylococcus aureus UCN18, which catalyzed the syn-
thesis solithromycin (3) using an azide-functionalized
Compounds with greater potency will have a higher re-
ciprocal MIC value (i.e., 1/MIC). Thus, a linear regression
plot was generated for mass count percent increase as a
function of 1/MIC. The strong correlation (R2 = 0.96) is
consistent with trends observed in in situ click chemistry
(Figure 5).6-8
macrolide precursor and 3-ethynylaniline (2) in cellulo
.
We extended our method to a library of twelve alkyne
fragments and observed a trend consistent with in situ
click chemistry wherein the most potent compounds as
measured by MIC were formed in greatest amount (i.e.,
mass percent increase). The formation of clicked triazole
products was confirmed by performing LC−MS analysis
and comparison with authentic material prepared inde-
pendently by Cu(I) catalysis using both retention time and
m/z values. We are confident this novel methodology will
find utility in the field of antibiotic drug discovery, particu-
larly campaigns focused on addressing the threat of bacte-
rial resistance.
In principle, in cellulo click chemistry could be applied in
a multicomponent fashion, though our initial trials with
multiple alkynes didn’t show a strong correlation between
MIC and mass count percent increase. We attribute this
phenomenon to a marked increase in system complexity
wherein differential rates of alkyne diffusion and attendant
concentrations result in inconsistent data. The visual ob-
servation of bacterial inhibition by ICCC using the MIC plat-
form can arise from three scenarios: (1) additive effects of
the two compounds; (2) synergistic effects of the two
compounds; or, (3) formation of the clicked triazole. Ulti-
mate confirmation of triazole formation by ICCC comes
from LC−MS analysis of the reaction mixture and compari-
son with an authentic sample to ensure matching retention
time and m/z values. In addition, the results obtained
herein with in cellulo synthesis of solithromycin (3) match
those reported in situ click using bacterial ribosomes and
ribosomal subunits. In other words, fragments for in situ
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Protein Structure Enables Target-Guided Synthesis of a Potent In-
Cell Inhibitor of Botulinum Neurotoxin. Angew. Chem. Int. Ed.
2015, 54, 7114-7119.
(16) Deyle, K. M.; Farrow, B.; Hee, Y. Q.; Work, J.; Wong, M.; Lai,
B.; Umeda, A.; Millward, S. W.; Nag, A.; Das, S. A protein-targeting
strategy used to develop a selective inhibitor of the E17K point
ASSOCIATED CONTENT
Supporting Information
Experimental procedures and characterization of new com-
pounds. The Supporting Information is available free of
charge on the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
,
* randrade@temple.edu
Author Contributions
The manuscript was written through contributions of all au-
thors.
ACKNOWLEDGMENT
We thank Prof. Alexander Mankin (Univ. of Illinois at Chicago)
for kindly providing us with the Staphylococcus aureus
UCN18 strain. We thank Drs. Charles W. Ross III, Ian Glassford
and Christiana Teijaro for carrying out preliminary experi-
ments and assistance with LC−MS experiments. We thank Mr.
Yanfeng Fan for assistance resolving polar compounds. We
thank Dr. Charles DeBrosse, Director of the NMR Facility at
Temple Chemistry, for kind assistance with NMR experiments.
ABBREVIATIONS
mutation in the PH domain of Akt1. Nature chemistry 2015,
455-462.
7,
ICCC, in cellulo click chemistry; ISCC, in situ click chemistry;
bovine BHI, brain heart infusion; MIC, minimum inhibitory
concentration; bCAII, bovine carbonic anhydrase II; MRM,
multi reaction monitoring; AZY, azithromycin; DMSO, dime-
thyl sulfoxide.
(17) Glassford, I.; Teijaro, C. N.; Daher, S. S.; Weil, A.; Small, M.
C.; Redhu, S. K.; Colussi, D. J.; Jacobson, M. A.; Childers, W. E.;
Buttaro, B.; Nicholson, A. W.; MacKerell, A. D.; Cooperman, B. S.;
Andrade, R. B. Ribosome-Templated Azide-Alkyne Cycloadditions:
Synthesis of Potent Macrolide Antibiotics by In Situ Click
Chemistry. J. Am. Chem. Soc. 2016, 138, 3136-3144.
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