Expanded therapeutic potential in activity space of next-generation 5-nitroimidazole antimicrobials with broad structural diversity

Article abstract of DOI:10.1073/pnas.1302664110

Metronidazole and other 5-nitroimidazoles (5-NI) are among the most effective antimicrobials available against many important anaerobic pathogens, but evolving resistance is threatening their long-term clinical utility. The common 5-NIs were developed decades ago, yet little 5-NI drug development has since taken place, leaving the true potential of this important drug class unexplored. Here we report on a unique approach to the modular synthesis of diversified 5-NIs for broad exploration of their antimicrobial potential. Many of the more than 650 synthesized compounds, carrying structurally diverse functional groups, have vastly improved activity against a range of microbes, including the pathogenic protozoa Giardia lamblia and Trichomonas vaginalis, and the bacterial pathogens Helicobacter pylori, Clostridium difficile, and Bacteroides fragilis. Furthermore, they can overcome different forms of drug resistance, and are active and nontoxic in animal infection models. These findings provide impetus to the development of structurally diverse, next-generation 5-NI drugs as agents in the antimicrobial armamentarium, thus ensuring their future viability as primary therapeutic agents against many clinically important infections.

Full text of DOI:10.1073/pnas.1302664110

Expanded therapeutic potential in activity space of
next-generation 5-nitroimidazole antimicrobials with
broad structural diversity
Yukiko Miyamotoa, Jarosław Kalisiakb, Keith Korthalsb, Tineke Lauwaetc, Dae Young Cheunga, Ricardo Lozanoa,
Eduardo R. Coboa, Peter Upcroftd, Jacqueline A. Upcroftd, Douglas E. Berga, Frances D. Gillinc, Valery V. Fokinb,
K. Barry Sharplessb, and Lars Eckmanna,1
Departments of aMedicine and cPathology, University of California, San Diego, La Jolla, CA 92093; bDepartment of Chemistry and The Skaggs Institute for
Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; and dQueensland Institute of Medical Research, Brisbane, QLD 4006, Australia
Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved September 9, 2013 (received for review February 8, 2013)
specificity of 5-nitro drugs stems largely from the requirement
for low redox potential electron transfers that do not occur in
mammalian cells (3), although other, poorly defined aspects may
also be important (4).
Metronidazole and other 5-nitroimidazoles (5-NI) are among the
most effective antimicrobials available against many important
anaerobic pathogens, but evolving resistance is threatening their
long-term clinical utility. The common 5-NIs were developed de-
cades ago, yet little 5-NI drug development has since taken
place, leaving the true potential of this important drug class
unexplored. Here we report on a unique approach to the modular
synthesis of diversified 5-NIs for broad exploration of their anti-
microbial potential. Many of the more than 650 synthesized com-
pounds, carrying structurally diverse functional groups, have
vastly improved activity against a range of microbes, including
the pathogenic protozoa Giardia lamblia and Trichomonas vagina-
lis, and the bacterial pathogens Helicobacter pylori, Clostridium
difficile, and Bacteroides fragilis. Furthermore, they can over-
come different forms of drug resistance, and are active and non-
toxic in animal infection models. These findings provide impetus
to the development of structurally diverse, next-generation 5-NI
drugs as agents in the antimicrobial armamentarium, thus ensur-
ing their future viability as primary therapeutic agents against
many clinically important infections.
Antimicrobial therapy with Mz is usually effective, with re-
ported success rates of 70–99%, depending on the specific infec-
tious agent and patient population (5). However, Mz treatment
failure and resistance occur for all target pathogens. For ex-
ample, >50% of H. pylori cases are resistant to Mz in some
developing countries (6). As many as 10–20% of patients with
giardiasis show clinical resistance to Mz (7), while 2–4% of
clinical T. vaginalis isolates display varying degrees of Mz re-
sistance (8). In some cases, Mz resistance can be overcome by
treatment with other 5-NI drugs, but many resistant microbial
strains exhibit cross-resistance between the major currently avail-
able 5-NI drugs (9). Multiple mechanisms have been implicated
in 5-NI drug resistance, including a diminished capacity to re-
duce and activate 5-nitro prodrugs (10, 11) and detoxification
of nitro drug radicals (12).
The common 5-NI drugs have different simple side chains at
the 1-position of the imidazole, e.g., Mz possesses a hydroxyethyl
group (Fig. 1A) and tinidazole has an ethylsulfonylethyl
group. These modifications mostly affect the pharmacokinetic
properties of the drugs but have only limited influence on drug
potency or ability to overcome resistance (9). However, other
structural modifications of 5-NI compounds can improve anti-
microbial activity and resistance profiles (4, 13) or confer new
antimicrobial activities, as shown for the kinetoplastid Trypanosoma
infectious diseases antibiotics medicinal chemistry
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ntibiotics are among the greatest advances in medicine, yet
their utility is constantly threatened by the development of
A
resistance due to the high genetic adaptability of many target
microbes. Most common antibiotics belong to a small number of
functional and structural classes that target a limited set of mi-
crobial processes, including cell wall synthesis, protein translation,
DNA replication, RNA transcription, and unique metabolic
pathways. Despite these seemingly limited targeting opportunities,
improved compounds have been developed within specific anti-
biotics classes over several drug generations with expanded po-
tency and microbial range, as best illustrated by next-generation
β-lactam antibiotics (1).
Significance
Drugs against disease-causing microbes are among the major
achievements of modern medicine, but many microbes show
a tenacious ability to develop resistance, so they are no longer
killed by available drugs. We show here for an important class
of these drugs, represented by the common drug metronida-
zole, that broad modifications of the basic drug structure can
improve drug activities against several clinically important
microbes and unexpectedly overcome different forms of re-
sistance. Several of these new drugs cure infections in animal
models and are safe in initial toxicity evaluations. These find-
ings provide reasons to develop this class of drugs as human
medicines in the ongoing fight against disease-causing microbes.
Of particular importance among antibiotics are 5-nitro drugs,
characterized by a nitro functional group in the 5-position of
a five-membered heterocycle (imidazole, thiazole, or furan). The
prototype and most commonly used drug of this class is the
5-nitroimidazole (5-NI) compound, metronidazole (Mz). Listed
as an essential medicine by the World Health Organization, it is
one of the most versatile antibiotics in clinical practice, targeting
a wide range of anaerobic microbes from protozoa, including
Giardia lamblia, Trichomononas vaginalis, and Entamoeba histo-
lytica, to bacteria, such as Helicobacter pylori, Clostridium difficile,
and Bacteroides fragilis (2).
Mz and other 5-nitro antimicrobials are prodrugs that must be
activated by reduction in the target microbe to generate toxic,
short-lived radical intermediates. The radicals form adducts with
different microbial molecules, including DNA, proteins, and
lipids, although the exact molecular targets and specific func-
tional consequences are incompletely understood. The microbial
Author contributions: Y.M., J.K., V.V.F., K.B.S., and L.E. designed research; Y.M., J.K., K.K.,
D.Y.C., R.L., and E.R.C. performed research; J.K., K.K., P.U., J.A.U., D.E.B., F.D.G., V.V.F.,
K.B.S., and L.E. contributed new reagents/analytic tools; Y.M., T.L., and L.E. analyzed data;
and Y.M., J.K., J.A.U., D.E.B., F.D.G., V.V.F., and L.E. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: leckmann@ucsd.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1302664110/-/DCSupplemental.
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cruzi (14). Despite these promising observations, the full antimi-
crobial potential of 5-NI drugs is not known, partly because com-
mercial drug development largely ceased after approval of the
first-generation 5-NI drugs beginning in the 1960s. Concerns
about long-term safety may have contributed to this situation,
but extensive clinical studies have shown that these compounds
are safe and have no relevant long-term toxicity in humans. For
example, a follow-up of 771 women treated with Mz 10 or more
years previously revealed no excess cancer risk (15).
The goal of the present studies was to generate a large library
of structurally diverse 5-NI compounds for comprehensive and
unbiased evaluation of the therapeutic potential of this impor-
tant class of antimicrobials. Using a unique modular approach to
5-NI synthesis, we show here that many of the >650 synthesized
5-NI compounds have vastly improved activity against a range of
microbial pathogens, as they display marked improvements in
broad-spectrum activity, can overcome different forms of 5-NI
drug resistance, and are active and nontoxic in animal infection
models. These findings substantially broaden the potential struc-
tural space suitable for further development of alternative nitro
antimicrobials for ultimate clinical use, and strongly suggest that
the systematic development of next-generation 5-NI drugs can
lead to superior agents in the armamentarium of antimicrobials
against a wide range of clinically important infections.
practically quantitative under simple, mild, and safe reaction
conditions, leaving few offensive by-products (17). The reaction
is also regiospecific and readily scalable (17), allowing rapid
production of substantial quantities for preclinical efficacy and
safety testing.
We first synthesized six azido derivatives of 5-NI, in which one
of the three available positions in the imidazole ring was func-
tionalized with azidoalkyl groups (Fig. 1B). The distance be-
tween the azido group and the imidazole core was minimized to
limit structural restrictions on the products of the subsequent
cycloaddition, although direct linkage of the azido group to the
imidazole core proved impossible, presumably because it com-
promised the stability of the heterocycle. We also generated
1-azidoalkyl imidazole compounds with conjugated furan- or
imidazole-based side chain in the 2-position (cores E and F,
Fig. 1B), as our previous studies had suggested that these mod-
ifications can yield potent antimicrobials (4).
We next assembled a library of 63 structurally diverse alkynes
as reaction partners for the azido cores (SI Appendix, Table S1).
The alkynes, which were obtained commercially or synthesized
de novo, were selected to achieve maximal structural diversity
with little consideration of conventional pharmacological criteria
such as hydrophobicity, electrochemical properties, or previously
successful side chains. We then performed all possible copper(I)-
catalyzed cycloadditions of the six azido-imidazole cores and 63
alkynes to synthesize 378 5-NIs (Fig. 1C and SI Appendix, Table
S1). Initial studies showed similar antimicrobial activities in
crude reaction mixtures compared with purified compounds,
indicating that the reaction mixtures could be rapidly screened
for activity without the time-consuming isolation of the products.
Results
Click Chemistry for Broad Structural Diversification of 5-NI Compounds.
Mz has an imidazole core substituted with 5-nitro and 1-hydrox-
yethyl groups (Fig. 1A). The nitro group is critical for antimi-
crobial activity, whereas the 3-position cannot be functionalized
without fundamentally altering the core heterocycle, leaving the
1-, 2-, and 4-positions for potential modifications to improve
activity. Prior studies on side chain modifications had not re-
vealed any clear structural themes that could be exploited
to generate compounds with superior activity (4, 13). As an
alternative, we reasoned that generating broad unbiased struc-
tural diversity might be more powerful than incremental opti-
mization for developing improved antimicrobials. Therefore,
we designed a synthetic approach for exponentially leveraging
the diversity of two reaction partners to generate a broad range
of 5-NI derivatives. The chemistry exploits the copper(I)-cata-
lyzed azide alkyne cycloaddition (“click reaction”), in which an
azide and an alkyne are united to yield a 1,4-substituted 1,2,3-
triazole (16). This reaction is particularly suitable for generat-
ing libraries for antimicrobial drug development, as yields are
Broad Antimicrobial Activities of Diverse 5-NI Compounds. To test
the antimicrobial activity of the 5-NI library, we used a range of
clinically important protozoa and bacteria that are commonly
treated with Mz. The intestinal parasite, G. lamblia, is a major
worldwide protozoal cause of diarrheal disease, T. vaginalis is
a common protozoal cause of sexually transmitted disease of the
genitourinay tract, and H. pylori and C. difficile are major bac-
terial causes of infectious disease in stomach and colon, re-
spectively. Using quantitative growth and survival assays, we
found that 66% of the 378 tested compounds had supe-
rior activity relative to Mz against G. lamblia and 40% against
T. vaginalis, with up to 50- to 500-fold lower EC50 relative to
Mz (Fig. 2A and SI Appendix, Tables S2 and S3). Marked activity
improvements were also observed for the bacterial pathogens:
40% of the compounds were superior to Mz against H. pylori
and 25% against C. difficile (Fig. 2A and SI Appendix, Table S4).
Some compounds displayed highly selective improvement
in activity against one of the target microbes, whereas others
exhibited greater activity against more than one microbe (Fig. 2B
and SI Appendix, Tables S2−S4). Analysis of the relationships
between different antimicrobial activities revealed modest but
significant positive correlations between the protozoa (r = 0.38,
P < 0.001) and bacteria (r = 0.53, P < 0.001) (Fig. 2C and SI
Appendix, Fig. S1). Moreover, superior antibacterial activity
correlated with improved antiprotozoal activity, and conversely,
superior antiprotozoal activity correlated with better antibacte-
rial activity (Fig. 2C and SI Appendix, Fig. S1). For example, 44
of 58 (76%) of the compounds that were more active than Mz
against both bacterial pathogens were also more active against
both protozoa. Together, 12% (n = 44) of the 378 tested com-
pounds were superior to Mz against all four pathogens (Fig. 2C).
This percentage of broadly active compounds significantly
exceeded (by >fourfold) the predicted 2.6% of compounds if
improvements in activity against each individual microbe were
entirely independent from improvement against the other
microbes, as calculated by multiplying the individual percentages
of superior compounds for each of the four target pathogens
(i.e., 66%, 40%, 40%, and 25%; Fig. 2A).
Fig. 1. Synthesis of comprehensive new 5-NI library. (A) Structure of Mz. (B)
Six different 5-NI cores (A−F) were synthesized with a “clickable” azide (N3)
functional group. (C) Scheme of click chemistry-facilitated synthesis of 5-NI
triazole library.
Given the broad-spectrum activity of several compounds, we
questioned whether the nitro compounds were also active against
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Fig. 2. Expanded antimicrobial activity range of structurally diverse 5-NI compounds. (A) The activities of 378 compounds were tested against the indicated
protozoa and bacteria in 24- to 48-h growth assays using ATP levels or OD600 as readouts. Each data point or number represents the mean EC50 for one
compound, with Mz shown in blue for comparison. (B) Examples of compounds with enhanced broad-spectrum or pathogen-selective activity (key values in
bold). (C) Relationships between activities of individual compounds against the four target pathogens. Compounds that exceeded the activity of Mz (blue
point) for both bacteria (black circles in light pink-shaded region, Left) were examined for their activities against the two protozoa (Right). The region
containing compounds with superior activity against all four pathogens is shaded in salmon color. (D) The relationship between compound activities against
two colonic bacteria (Upper). The orange-shaded region highlights compounds more active than Mz against the opportunistic pathogen, C. difficile, but less
active than Mz against the commensal, B. fragilis. Assay sensitivity is depicted by the dashed green line. The table (Lower) lists examples of compounds that
showed greater or lesser selectivity than Mz against C. difficile compared with B. fragilis.
intestinal commensals, which might impact their clinical utility.
None of the 378 nitro compounds exhibited activity against the
commensal Escherichia coli (strain K12) up to a maximum con-
centration of 100 μM, suggesting that the newly synthesized 5-NI
drugs, like Mz, do not have nonspecific toxicity in facultative
microbes naturally resistant to this drug class. B. fragilis is an
important commensal that normally resides in the intestinal lu-
men but can cause peritoneal infections when translocated due
to gut perforations. A substantial fraction (20%) of compounds
had superior activity against B. fragilis compared with Mz (Fig. 2
A and B and SI Appendix, Table S4). Importantly, comparison of
activities against B. fragilis and C. difficile, both of which colonize
the colon, revealed a range of ratios (Fig. 2D), indicating that
some nitro drugs exist with improved relative selectivity against
the opportunistic pathogen C. difficile compared with the com-
mensal B. fragilis.
Collectively, these results indicate that many of the synthe-
sized 5-NIs have a marked, and for some highly selective,
increase in activity against each of the individual microbes.
Furthermore, a significantly greater than expected number of
compounds were superior to Mz against multiple different
microbes, making these compounds strong candidates for broad-
spectrum antibiotics. Importantly, the vast majority of the com-
pounds (362/378, >95%) had no cytotoxicity in human HeLa
cells and many showed therapeutic indices equal or superior to
Mz (SI Appendix, Table S5).
Beyond natural variability in antimicrobial susceptibility, re-
sistance to Mz can develop during antimicrobial therapy, and Mz
resistance can be readily induced in bacteria and protozoa in the
laboratory (9). Importantly, currently approved 5-NI drugs ex-
hibit cross-resistance to Mz and show similar clinical failure rates
(9). To determine whether the 5-NI compounds can overcome
acquired Mz resistance, we used two independently derived
syngeneic Mz-resistant (MzR) lines of G. lamblia and two clinical
MzR isolates of T. vaginalis. Remarkably, all of the 378 tested
compounds exhibited activity against the two MzR Giardia lines
that was superior (i.e., lower EC50) to Mz (Fig. 3A, light pink
shading; SI Appendix, Table S2). Moreover, 23% of the com-
pounds (n = 87) were also more active against both MzR lines
than Mz was against the corresponding parental Mz-sensitive
(MzS) lines (Fig. 3A, salmon-colored shading). Thus, these 87
compounds effectively overcame acquired Mz resistance in
Giardia, as they could kill MzR lines at least as effectively as Mz
could kill MzS lines.
For T. vaginalis, 176 of 377 tested compounds (47%) were
superior to Mz in two unrelated MzR isolates (Fig. 3A, light pink
shading; SI Appendix, Table S3). Of these, 10 compounds (2.6%
of all compounds) exhibited activity against both MzR lines that
exceeded Mz activity against the MzS lines (Fig. 3A, salmon-
colored shading). Thus, the range of activities, if not the fre-
quency of compounds with superior activity, against T. vaginalis
was similar to G. lamblia, supporting the conclusion that our
comprehensive 5-NI drug library contains compounds with di-
verse activities against a wide range of microbes and forms of
drug resistance.
As a further test of the ability to overcome nitro drug re-
sistance, we used a double mutant of H. pylori for two reductases,
NADPH flavin oxidoreductase (FrxA) and oxygen-insensitive
NADPH nitroreductase (RdxA), that activate nitro drugs and
are involved in clinical drug resistance (18). Screening of the
library revealed that 98.6% of compounds lost activity against
the double mutant (Fig. 3B and SI Appendix, Table S4), which
demonstrates that reduction by one or both of these reductases is
critical for compound activity and further underlines the high
molecular specificity of the 5-NI compounds. Interestingly, we
also found five compounds that remained active in the 10- to 20-
μM range in the double mutant. One of the compounds was also
toxic in HeLa cells, but the other four had no apparent cyto-
toxicity in these cells (and no activity against E. coli up to 100
μM), suggesting that they retain microbial specificity and raising
the possibility that alternative reductive pathways may exist in
H. pylori to activate these nitro compounds.
Structurally Diverse 5-NI Compounds Can Overcome Different Forms of
Mz Resistance. Natural variability in antimicrobial susceptibility is
an important consideration in drug development. To address this
issue, we tested additional isolates of three of the target pathogens
and related the data to the initial screens. Activities showed
generally good correlations (with r values of 0.896, 0.695, and
0.388 for Giardia, Trichomonas, and Helicobacter, re-
spectively; P < 0.01 for all three; SI Appendix, Fig. S2 and
Tables S2−S4), but differences were apparent between the
pathogens. For Giardia, 63% of the compounds exhibited
superior activity over Mz against both clinical isolates, whereas
this percentage was lower for T. vaginalis (21%) and H. pylori
(26%), underlining the importance of testing a range of isolates
to avoid pursuit of compounds with limited, isolate-specific ac-
tivity. Nonetheless, these data demonstrate that the 5-NI library
had multiple compounds with superior activities against different
clinical isolates of the target pathogens.
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Fig. 3. New 5-NI compounds overcome Mz re-
sistance. (A) Compounds were tested against MzR
strains of G. lamblia (Left) and T. vaginalis (Right),
with those more active than Mz (purple) highlighted
by light pink boxes. Of these, compounds more ac-
tive against both MzR lines than Mz against the
respective MzS lines (light blue) are further high-
lighted by salmon-colored boxes. (B) Activities against
wild-type and double mutant (ΔfrxA ΔrdxA) strains of
H. pylori. Five compounds (red dots) had measurable
activity against the mutant; all others were below the
assay sensitivity (dashed green line, Upper). Examples
of active compounds are listed in the table (Lower).
Distinct and Predictive Bioactivity Landscapes of 5-NI Compounds. To
begin to understand the relationship between compound struc-
tures and antimicrobial activities, we used principal component
analysis to visualize the 5-NI compounds in a structural space
and then highlighted the most active compounds within that
space. The top 10% compounds with broad-spectrum activity
against all four target pathogens were located in different regions
within the space (Fig. 4A). By comparison, localization of the
most active compounds against each of the individual pathogens
revealed distinct patterns (“activity fingerprints”) for the four
pathogens (SI Appendix, Fig. S3), which overlapped only partly
with the pattern of broad-spectrum compounds. These data
suggest that the structural requirements for activity improvement
heavily depend on the particular microbes, but also show that
broad-spectrum activity can be achieved in multiple structural
domains.
Further evaluation of the structure−activity relationships by
principal component analysis for one of the pathogens, MzR
G. lamblia, revealed a nonrandom distribution of bioactivity in the
chemical space occupied by the compounds, with distinct activity
peaks and valleys (Fig. 4B). Based on this observation, we ap-
plied machine learning tools to construct a mathematical model
for bioactivity prediction. Although decision tree analysis yielded
excellent (84%) cross-validation in the set of 378 training com-
pounds, even better predictability (87%) was achieved with
a service vector machine approach. To test the predictive power
of the model, we synthesized another set of 281 test compounds
by click chemistry using the same six 5-NI cores A−F (Fig. 1B)
but 47 different alkynes (SI Appendix, Table S6). These alkynes
were structurally distinct from those used in the initial library,
but occupied an overlapping chemical space (SI Appendix, Fig.
S4). The model was used to divide the test compounds into two
groups with predicted superior or inferior activity in MzR cells
relative to Mz in the parental MzS Giardia cells. Some 82% of
predicted superior compounds exhibited superior activity in an-
timicrobial testing, while 90% of predicted inferior compounds
had inferior activity in the tests (Fig. 4C and SI Appendix, Table
S7). Together, bioactivity of 86% (248 of 281) of the test com-
pounds against MzR G. lamblia was correctly predicted, in-
dicating the excellent utility of the predictive model. The findings
experimentally validate prior contentions that structural analysis
by chemical descriptors can be exploited to define chemical
spaces with promising structure−activity relationships (19, 20).
As for the training compounds, the vast majority of test com-
pounds (260/279, 93%) had no measurable cytotoxicity in human
HeLa cells (SI Appendix, Table S8).
Structural Determinants of Superior 5-NI Activity. To define the
structural determinants of superior antimicrobial activity of the
5-NI triazole compounds, we first examined the importance of
the azido-imidazole core. Compounds based on cores A−C had
similar average activities against MzS and MzR Giardia (Fig.
5A). Core D compounds had low or no activity against either,
whereas the E series compounds had, on average, significantly
Fig. 4. Predictive bioactivity landscape of 5-NI compounds. A structural space was generated by principal component analysis using activity data of the 378
compounds in the 5-NI library against the four target microbes. (A) The individual compounds were plotted in the resulting space with the top 10% most
potent broad-spectrum compounds shown as red circles. (B) A structural space was constructed from the activity data against MzR G. lamblia. Activities of all
compounds were plotted in the space (z axis), and a contour graph was generated using the indicated color scale. (C) A service vector machine model was
constructed from the activity data of the 378 compounds against MzR Giardia (training set) and applied prospectively to a new set of 281 independently
synthesized 5-NI compounds (test set). Correctly predicted compounds, as defined by coincidence of model prediction and assay-determined activity against
the two MzR Giardia lines, are highlighted by background coloring (light pink and light blue) for predicted superior (Left) and inferior (Right) compounds.
Each data point represents one compound. The light blue dots show Mz activity against the MzS parental lines for comparison.
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not observed over the 3-d treatment course. In vivo efficacy
could not be explained by greater in vitro antigiardial activity or
altered aqueous solubility (Fig. 6B). However, active compounds
had higher serum levels than inactive compounds by 2 h after
oral administration (Fig. 6B), suggesting that systemic bio-
availability was important for antigiardial efficacy. Examination
of the structure−activity relationships revealed five chemical
attributes, different from those in the Lipinski rules commonly
used to predict bioavailability of new compounds (21), that could
effectively differentiate in vivo active and inactive compounds in
the chemical space of all library compounds (SI Appendix, Figs.
S5 and S6). Examples of active compounds show that promising
activities against giardiasis can be found among very different
chemical structures (Fig. 6C). Initial evaluation of the toxicity
profile of several of these compounds by micronucleus assay with
mammalian CHO cells using current Organization for Economic
Cooperation and Development guidelines revealed no acute
genotoxicity (SI Appendix, Fig. S7). Furthermore, oral adminis-
tration of five high doses (100 mg/kg; equivalent to ∼10 × ef-
fective dose) to adult mice over 3 d revealed no toxicity, as
assessed by liver enzyme panels and histological and hemato-
logical evaluation.
Discussion
Our results indicate that next-generation 5-NI drug candidates
can be synthesized and rapidly tested in vitro and in vivo by
combining the ease of modular click chemistry with cell culture
assays, predictive machine learning tools, and animal models.
Thus, it is readily feasible to advance from initial synthesis of
hundreds of compounds to focused animal testing in mere weeks,
thereby significantly shortening the initial development cycle in
drug design (17). This acceleration could promote a timely and
cost-effective drug development response to the emergence of
antimicrobial resistance that threatens public health. Adequate
preclinical safety testing will be needed before commencing
human trials, but even this step can be significantly shortened
and made more cost-effective by rapid production of kilogram
quantities of new drug candidates by click chemistry or other
chemistries suitable for combinatorial synthesis and ready side
chain preservation.
The observed increases in antimicrobial activity were striking
(>100-fold) for several of the 5-NI compounds, although the
underlying reasons remain to be established. Despite the wide-
spread use of Mz and other 5-NI drugs, their mechanisms of
antimicrobial killing remain incompletely understood. Reduction
of the prodrugs is required for activity, so higher affinity or faster
reaction kinetics of more potent compounds with the relevant
reductases may be important. The stability or molecular tar-
geting efficiency of activated nitro drug radicals may be more
favorable for the new compounds (22), although chemical re-
duction with dithionite followed by immediate antimicrobial
testing led to complete activity loss, suggesting that the toxic
products formed by reduction are not very stable under these
conditions. Alternatively, the spectrum of nitro drug adduction
targets may be different for the most potent compounds (4, 23).
In any case, the observation that many compounds showed im-
proved broad-spectrum activity would suggest that the un-
derlying mechanisms are likely to entail broadly relevant drug
properties, such as improved membrane permeability, that are
not limited to particular molecular targets or microbial features.
A key observation of our studies is that many new compounds
could effectively overcome nitro drug resistance in different
microbes. Mz resistance is functionally heterogeneous, with sev-
eral causative mechanisms for each of the target microbes and
between different microbes (7, 24). For example, resistance in
Giardia involves down-regulation of nitro drug-activating sys-
tems, including different reductases and redox proteins and
metabolites (11, 25), whereas Trichomonas also produces re-
sistance proteins that directly detoxify nitro drugs (12) and
Bacteroides has transporters that can remove nitro drugs from
the cell (26). Despite this mechanistic diversity, for each of the
Fig. 5. Structure−activity relationships of 5-NI building blocks. The entire
library of 659 5-NI compounds (composed of the 378 training and 281 test
compounds) was examined for structure−activity relationships of the two
building blocks, azido-5-NI and alkyne, used for the click chemistry-facili-
tated synthesis. (A) The influence of the azido-5-NI cores (A−F, Fig. 1B) on
activity against Mz-sensitive (MzS, Left) and Mz-resistant (MzR, Right) Giar-
dia, with average activities shown as red lines. (B) Data of all compounds
(cpds) generated from cores A−C (to minimize core bias in the alkyne eval-
uation) in a structural space derived by principal component analysis. The
top most active compounds against MzS and MzR Giardia are highlighted in
light brown and red, respectively. Several regions with clustering of the most
active compounds are boxed, and the corresponding alkynes used for com-
pound synthesis are depicted.
higher activities than those of the other cores against both MzS
and MzR Giardia (Fig. 5A). To explore the contribution of the
alkyne partners to triazole bioactivity, we focused on the anti-
giardial activity of A−C series compounds to minimize core bias.
Principal component analysis revealed a nonrandom, patchy
distribution of the superior triazoles, occupying several distinct
areas in the chemical space of the entire group of core A−C
compounds (Fig. 5B). The corresponding alkynes in the active
regions showed marked structural similarities within each of
these regions, but little between different regions. These results
indicate that several structurally unrelated groups of compounds
can yield superior antigiardials.
In Vivo Efficacy of 5-NI Triazoles in Animal Infection Models. Because
many 5-NI compounds exhibited promising antimicrobial activ-
ities in vitro, we next examined their in vivo efficacy in a murine
model of giardiasis. Out of 16 structurally representative com-
pounds, 8 (50%) displayed significant activity against giardiasis,
and 7 (45%) were more efficacious at clearing Giardia than Mz
at a standard dose of 10 mg/kg (Fig. 6A, red). Acute toxicity was
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Fig. 6. In vivo efficacy of 5-NI compounds against giardiasis. (A) Adult C57BL/6 mice were orally infected with G. lamblia and given five 10 mg/kg oral doses of
the indicated compounds over 3 d at 12-h intervals, and trophozoite numbers in the small intestine were determined. The dashed green line represents the
assay sensitivity. Data are mean + SEM (n ≥ 5); *P < 0.05 vs. vehicle-treated controls. Compounds significantly more efficacious than Mz (light blue) are
highlighted in red. (B) The relationships of in vivo bioactivity, in vitro activity (Left), aqueous solubility (Center), and measured serum drug concentrations
(Right, green dashed line represents the assay sensitivity; Mz is shown in blue for comparison). (C) Examples for in vivo active compounds, along with their in
vitro activities (Table in Lower Right) against MzS and MzR Giardia.
resistant microbes, we were able to identify nitro compounds that
could combat resistance, and some compounds were broadly
effective against different resistant microbes. Several basic
explanations, alone or in combination, may account for this
finding. New nitro compounds may be activated by alternative
pathways that are not involved in Mz resistance, or they may
bypass Mz detoxification or efflux pathways. Alternatively, im-
proved nitro compounds may be activated by the same pathways
that are suppressed in Mz resistance, but the residual activity of
these pathways, which may not be completely shut down due to
significant fitness costs (9), is sufficient to mediate adequate
activation of the most potent nitro compounds.
Our data suggest that incremental structural modifications, as
frequently used for lead compound optimization (27), may not
be an optimal strategy for 5-NI drugs, as compounds with dif-
ferent and unpredictable structural features had desirable anti-
microbial properties in vitro and in vivo. Although the precise
mechanistic reasons for this counterintuitive observation remain
to be determined, 5-NIs can be activated by different microbial
pathways and probably target multiple microbial molecules (11,
12, 23) whose relative importance may depend on the compound
and microbe. Because the specific target molecules of 5-NI drugs
are not fully understood (23), a strategy focused on achieving
effective whole-cell antimicrobial activity in the relevant systems
may be the best choice for making rapid progress in developing
improved antimicrobials in the clinically indispensable 5-NI class.
Materials and Methods
Azido-alkyl-5NI heterocycles and 60 alkynes were synthesized de novo, and 50
additional alkynes were purchased. Click reactions were performed with
azides and alkynes in t-BuOH:H2O with sodium ascorbate and CuSO4. In vitro
activities (EC50) were determined with single-step ATP assay or OD600 after
24–48 h growth in culture. Structure–activity relationships were analyzed
with chemical descriptors (E-Dragon 1.0) and machine learning software
(WEKA). In vivo efficacy was assessed in C57BL/6 mice infected with
G. lamblia GS/M. See SI Appendix for details.
ACKNOWLEDGMENTS. We thank Elaine Hanson, Lucia Hall, and Christina
Yoon for technical assistance. This work was supported by National Institutes
of Health Grants AI075527, DK035108, and DK080506.
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