Optimization of methionyl tRNA-synthetase inhibitors for treatment of Cryptosporidium infection

Article abstract of DOI:10.1128/AAC.02061-18

Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most need

Full text of DOI:10.1128/AAC.02061-18

AAC Accepted Manuscript Posted Online 11 February 2019
Antimicrob. Agents Chemother. doi:10.1128/AAC.02061-18
Copyright © 2019 American Society for Microbiology. All Rights Reserved.
1
2
3
4
Optimization of methionyl tRNA-synthetase inhibitors for treatment of
Cryptosporidium infection
5
a*
a
a
b
6
7
8
9
0
Frederick S. Buckner , Ranae M. Ranade , J. Robert Gillespie , Sayaka Shibata ,
a
b
b
a
Matthew A. Hulverson , Zhongsheng Zhang , Wenlin Huang , Ryan Choi , Christophe
b
b
c
c
d
L. M. J. Verlinde , Wim G. J. Hol , Atsuko Ochida , Yuichiro Akao , Robert K.M. Choy
Wesley C. Van Voorhis , Sam L.M. Arnold , Rajiv S. Jumani , Christopher D. Huston ,
Erkang Fanb*
a
a
e
e
1
1
1
a
b
1
1
1
1
2
3
4
5
Affiliations: Department of Medicine and Department of Biochemistry , University of
c
Washington, Seattle, Washington, USA; Takeda Pharmaceuticals , Osaka, Japan;
Drug Development Program , PATH, San Francisco, CA, USA; Department of
Medicine , University of Vermont, Burlington, Vermont, USA.
d
e
1
1
1
1
6
7
8
9
* Corresponding authors:
Frederick S. Buckner: fbuckner@uw.edu
Erkang Fan: erkang@uw.edu

2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
ABSTRACT: Cryptosporidiosis is one of the leading causes of moderate to severe
diarrhea in children in low-resource settings. The therapeutic options for
cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor
activity in the most needy populations of malnourished children and HIV infected
persons. This paper describes the discovery and early optimization of a class of
imidazopyridine-containing compounds with potential for treating Cryptosporidium
infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase
(MetRS), an enzyme that is essential for protein synthesis. The most potent
compounds inhibited the enzyme with K values in the low picomolar range.
i
Cryptosporidium cells in culture were potently inhibited with EC50 values as low 7 nM
and >1000-fold selectivity over mammalian cells. A parasite persistence assay
indicates that the compounds act by a parasiticidal mechanism. Several compounds
were demonstrated to control infection in two murine models of cryptosporidiosis without
evidence of toxicity. Pharmacological and physicochemical characteristics of
compounds were investigated to determine properties that were associated with higher
efficacy. The results indicate that MetRS inhibitors are excellent candidates for
development for anti-cryptosporidiosis therapy.
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
INTRODUCTION:
Although the world has seen substantial progress in the reduction of child
mortality in recent decades, much work remains to achieve the United Nations’
Sustainable Development Goals (targeting 2030) for child survival (1). Diarrhea is one
of the leading causes of child mortality, responsible for 8% of deaths in children aged 1-
59 months (2). The Global Enteric Multicenter Study (GEMS) identified
Cryptosporidium to be the second leading cause of moderate to severe diarrhea in
young children at sites in Africa and Asia (3). The importance of cryptosporidiosis in
community diarrhea in developing countries was confirmed in the MAL-ED study (4).
Beyond the mortality risk, children who survive cryptosporidiosis suffer from growth and
developmental stunting which contribute to all-cause mortality and disability (5, 6). The
recent appreciation of the impact of cryptosporidiosis has drawn attention to the
inadequacies in the means to control this infectious disease. No vaccines are in clinical
use and the sole drug for treating cryptosporidiosis (nitazoxanide) has poor efficacy in
malnourished children and in patients with human immunodeficiency virus. Vaccine
development is likely to be slow due to difficulties raised by antigenic differences within
Cryptosporidium leading to poor cross protection between species and strains (7). New
anticryptosporidial drugs are likely to be the most rapidly developed technology to
address the burden of Cryptosporidium infection.
Recent studies suggest that Cryptosporidium is more closely related to gregarine
protozoa than to coccidians (8). The genus Cryptosporidium has 27 species that have
been identified worldwide that infect four classes of vertebrates (9). The species

6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
primarily responsible for human cryptosporidiosis are C. hominis and C. parvum. Water-
and foodborne transmission are the major modes of infection, although person to
person contact is also described (9). The small intestine is the primary site of
Cryptosporidium infection in humans, although extra-intestinal sites such as the biliary
tract, lungs, and pancreas can be involved in immune compromised and immune
competent individuals (9-11). The extra-intestinal locations may have important
implications for developing therapeutics that act at all sites of infection.
Cryptosporidium mainly resides in an unusual niche in the intestinal epithelium known
as the parasitophorous vacuole, which is insulated from both the intestinal lumen as
well as the host cytoplasm. Therefore, it is not entirely known whether anti-
Cryptosporidium drugs should be optimized for luminal or plasma exposure, although a
recent paper emphasizes the importance of gastrointestinal (luminal) exposure in a
murine model (12).
Protein synthesis is a classic antimicrobial drug target, dating back to many of
the first antibiotics—such as chloramphenicol, tetracycline and erythromycin—that
inhibit bacterial protein synthesis. Recently, aminoacyl-tRNA synthetase (aaRS)
inhibitors have emerged as promising therapeutic candidates for targeting protein
synthesis. Using ATP hydrolysis, aaRSs catalyze the formation of tRNAs charged with
their cognate amino acids which serve as the substrates for the formation of new
peptides. Mupirocin, a small molecule inhibitor of isoleucyl-tRNA synthetase (13), has
been in clinical use for more than two decades as a topical treatment for
Staphylococcus infections. Tavaborole is a leucyl-tRNA synthetase inhibitor that was
approved by the FDA in 2014 for topical treatment of onychomycosis (14, 15).
Halofuginone, a prolyl-tRNA synthetase inhibitor (16), is approved for veterinary use
against Cryptosporidium in Europe, although a narrow therapeutic index makes it
unsuitable for human use. Three different aaRS inhibitors for systemic use are in clinical
trials, demonstrating the potential for safe use beyond topical applications. These
include the methionyl-tRNA synthetase (MetRS) inhibitor CRS3213 for Clostridium
difficile infections (ClinicalTrials.gov, NCT01551004), the leucyl-tRNA synthetase
inhibitor GSK2251052 for Gram-negative bacterial infections (17, 18), and the leucyl-
tRNA synthetase inhibitor GSK3936656 for multidrug resistant tuberculosis (19, 20).
Inhibitors of other aaRSs from protozoan parasites including Cryptosporidium,
Plasmodium, Trypanosoma and Toxoplasma are also in development (21, 22).
MetRS enzymes fall into two categories, MetRS1 and MetRS2 (23). C. parvum
and C. hominis contain a single MetRS gene that aligns with the MetRS1 category,
meaning it has close homology to the MetRS of S. aureus, Trypanosoma spp., and the
human mitochondrial MetRS. Our group has been developing inhibitors to type 1
MetRS that are shown to have potent activity against trypanosomes and Gram positive
bacteria, including activity in animal models (24-27). Supporting this work are
numerous crystal structures of the inhibitors bound to the Trypanosoma brucei MetRS

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
enzyme (28-30), which is 76% identical (19 of 25 residues) to the C. parvum/hominis
MetRS within the inhibitor binding pocket. In this paper, the C. parvum MetRS was
characterized and MetRS inhibitors were shown to be highly potent with K values as
i
low as 0.9 pM. Compound 2093 had the most potent in vitro activity against C. parvum,
and reduced Cryptosporidium infection to low levels in two murine models without
showing signs of toxicity. The physicochemical and pharmacological features
associated with in vivo activity are discussed. The research illustrates the potential of
target-based drug discovery to develop a novel therapeutic against a formidable
eukaryotic pathogen.
RESULTS
Amino acid sequence alignment. Cryptosporidium parvum (UniProt Q5CVN0) and
Cryptosporidium hominis (UPI0000452AB0) have a single MetRS gene in their
respective genomes. These sequences were compared to the well-characterized
Trypanosoma brucei MetRS by mapping them onto the crystal structures of TbMetRS
bound to inhibitors. Table 1 shows the amino acid residues of TbMetRS that form
binding pockets that are in direct proximity to MetRS inhibitors (28-30). The C. parvum
and C. hominis sequences are identical to each other over the 25 residues forming the
compound binding pockets. The Cryptosporidium sequences share 19 identical amino
acids with TbMetRS and 18 identical residues with the human mitochondrial MetRS
over this region. Only 14 of the 25 amino acids are identical between the
Cryptosporidium MetRS and the human cytoplasmic MetRS in this region.
Enzymology. The C. parvum MetRS enzyme was over-expressed in E. coli and
purified by nickel affinity chromatography followed by size exclusion chromatography
(Fig. S1, Supplementary Data). The activity was confirmed in the aminoacylation assay
which detects the esterification of radiolabeled methionine to the tRNA substrate (Fig.
S2, Supplementary Data). However, in order to measure the Michaelis-Menten
constants for the enzyme, the ATP:PP
explained (31). The K for the methionine substrate was comparable to that observed
in S. aureus (both in house and published) (Table 2), but about 4-fold higher than the
value for the human mitochondrial MetRS (71 vs. 18 µM). The K for the ATP substrate
i
exchange assay was employed as previously
m
m
was about 2-3 fold higher than that observed in S. aureus (both in house and published)
(Table 2), and about 10-fold higher than the value for the human mitochondrial MetRS
(1040 vs. 85 µM).
MetRS inhibitor activities. The initial MetRS inhibitors were synthesized to target
trypanosomatid parasites (24-26, 30). The activities of selected compounds on:
CpMetRS enzyme, C. parvum parasites cultured in HCT-8 cells, and mammalian cells
are shown in Tables 3 and 4. The MetRS inhibitors are characterized by two ring

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
systems tethered by a linker. Table 3 shows four compounds in which the linker is an
alkyl chain (“linear linker”). The aminoquinolone (1312) (24) and urea-containing
compounds (1433) (25) are weakly active on the CpMetRS enzyme and show little
activity on C. parvum cultures. The fluoro-imidazopyridine compounds (1614 and 1717)
(26) have moderate activity on both the enzyme and cultures. Next, a series of 13
compounds containing a ring in the linker region were tested (Table 4). The 1,3-
dihydro-2-oxo-imidazole ring was found to be the most active and thus variants of this
scaffold were further investigated. Compound 2093 is the overall most potent
i
compound with a K value on CpMetRS of 0.9 pM and an EC50 between 7 nM and 36
nM determined in independent laboratories using different readout techniques
(luciferase reporter vs. fluorescence microscopy) and different C. parvum strains (see
Methods). Importantly, 2093 has no cytotoxicity on mammalian cells at the highest (50
µM) concentration tested. Examining the SAR of this scaffold indicates the relative
potency at the R
1
position as follows: ethyl>CH
2
CF
2
H> propyl>H. At the R
2
position, Cl
the
is slightly better than F (compare 2093 to 2114 and 2067 to 2062). Finally, at R
3
methoxy substitution provides moderately greater potency than chloro (compare 2114 to
2062 or 2093 to 2067 or 2258 to 2138). None of the compounds exhibited substantial
cytotoxicity on either mammalian cell line tested (CRL-8155 or HepG2).
The correlation between enzyme inhibitory activity and C. parvum growth
inhibitory activity was plotted for all the compounds shown in Tables 3 and 4. A strong
2
correlation was observed (R = 0.91) (Figure 1).
The compounds were also tested for inhibitory activity of mitochondrial protein
synthesis in HepG2 cells (COX-1 EC50, Tables 3 and 4). The COX-1 gene is encoded
by the mitochondrial genome and expressed in the mitochondrial protein synthesis
pathway. The MetRS inhibitors showed a wide range of EC50 values from low
micromolar to low nanomolar concentrations. In general, compounds with potent
activity on C. parvum cultures also had potent activity on the COX-1 enzyme (e.g.,
2093). A parallel experiment was performed on the same samples to measure levels of
the nuclear encoded SDH-A protein that is imported into the mitochondrion. A reduction
in levels of this protein reflects general toxicity to the cells. Except for compound 1433,
all compounds exhibited SDH-A EC50 values >25 µM, indicating no general toxicity at
this concentration.
The activity of the most potent MetRS inhibitor, 2093, was tested against a panel
of three different C. parvum strains including a clinical isolate from dairy calves and a
C. hominis strain (Table S1, Supplementary data). Compound 2093 had EC50 values
ranging from 0.006 – 0.029 µM against C. parvum strains and of 0.015 µM against
C. hominis. A wide therapeutic index was also documented with CC50 values >25 µM
against 3 mammalian cell lines.
Next, a parasite persistence assay was performed with 2093 and control
compounds nitazoxanide and MMV665917 (Fig. 2). The persistence curve for 2093

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
closely resembles that of MMV665917 which is believed to have a parasiticidal
mechanism of anti-Cryptosporidium activity and is dissimilar to the curve for
nitazoxanide which is believed to have a parasitistatic mechanism (32).
Efficacy in two murine C. parvum infection models.
Compounds with the highest in vitro potency were selected for efficacy studies
employing two different mouse models.
NOD SCID Gamma (NSG) mouse model (32): Adult mice (n=4 per group)
5
received a challenge dose of 10 oocysts and were treated with study compounds from
day 6-10 post-infection. Parasites were quantified in stool by PCR on day 5 and day 11
post-infection showing that 2093 was associated with 98.6% reduction (P<0.05) of
parasites. Compound 2069 resulted in 83.6% reduction ((P<0.05), whereas compound
2067 had only an 8.9% reduction (not statistically significant) (Fig. S3, Expt 1). A follow
up experiment showed similar results for 2093 and about the same level of parasite
reduction with 2259 (Fig. S3, Expt 2, P<0.05 for both compounds).
IFN- knockout mouse model (33): Efficacy experiments were also performed
using adult IFN- knockout mice and a luciferase expressing C. parvum strain. Adult
3
mice (n=3 per group) received a challenge dose of 10 oocysts, and like above, were
treated with study compounds from day 6-10 post-infection. Pooled feces from each
group were collected daily and the parasite load was quantified by luminometry. Again,
2093 was found to be highly efficacious (>4 log10 drop in luminescence) at the oral dose
of 50 mg/kg BID (Fig 3A). Compound 2093 was also tested at lower doses of 50 mg/kg
once per day (Fig. 3B) and at 25 mg/kg twice per day (Fig. 3C), and resulted in ~3-log10
drop in fecal parasite levels in both experiments. At 20 mg/kg once per day, 2093 gave
~1-log10 drop in parasite levels (day 9) that then rebounded to control levels after the
treatment was completed (Fig. 3D). Compound 2114 and 2259 at 50 mg/kg BID gave
similar profiles to 2093 with a 3-4 log10 drop in stool parasite levels (Fig. 3E and 3F),
whereas compounds 2258 appeared less active (Fig. 3C). Other compounds with
modest anticryptosporidial activity (1-2 log drop in fecal parasite loads) were 2138 and
2139 (Figs. 3G). Finally, the following compounds had no demonstrable activity: 2207
at 50 mg/kg PO QD nor 2080, 2240 or 2242 given at 25 mg/kg PO BID (data not
shown). A control compound with strong anti-Cryptosporidium activity, the “bumped
kinase inhibitor” 1369 (33), was included in some experiments for reference (Figures 3F
and 3G).
In all experiments, the mice were weighed daily. Weight loss was consistently
observed in the mice treated with vehicle control. When weights drop below 20% of
baseline, mice were euthanized per protocol requirements. (The lines end when all
mice were culled). There was some variability between experiments in the time it took
for control mice to reach terminal weight (e.g., compare panel 3A to panel 3F). The
mice receiving treatments that led to reduced Cryptosporidium infection had relatively

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
stable weights over the course of the experiment. Once the compound dosing was
completed (day 11), weight gain was sometimes observed (e.g. panel 3A and 3G).
Except for compound 2258, the MetRS inhibitors shown in Fig. 3 were well-tolerated
and led to all mice surviving (without >20% weight loss) until the end of the experiment.
Physicochemical properties, metabolism, pharmacokinetic studies.
Physicochemical properties of the compounds were calculated or measured
(Table 5). The molecular weights for the ring-linker series are in the 400 – 500 g/mol
range. The calculated log P values range from 3.25 to 4.75. Solubility of 2093, 2114,
and 2259 (the more active compounds in the mouse efficacy model) was higher than for
2067 and 2069. Plasma protein binding is high for all of the tested compounds, ranging
from 95% to >99.9%, with the exception of 2240 with a plasma protein binding value of
87.7%. Permeability was assessed for a subset of compounds using the PAMPA
method and showed fairly similar permeability values (226-319 nm/sec) for the tested
compounds. For comparison, a highly permeable compound, propranolol, had a
permeability value of 600 nm/sec (pH 7.4) and a low permeability compound,
methylclothiazide, has a value of 30 nm/sec (pH 7.4). The permeability of the
compounds was also calculated by computer algorithm, and indicated that the predicted
permeability for MetRS inhibitors is more similar to propranolol (known to have high
permeability) than methylclothiazide (known to have low permeability). The relationship
between the described chemical properties (i.e., the predicted permeability and the
solubility/EC50) and in vivo efficacy of the compounds was graphed (Fig. 4). This shows
that the most active compounds in vivo cluster at an intermediate level of permeability
and have a high ratio of solubility/EC50
.
In vitro measurements of liver microsome stability showed half-lives ranging from
2-29 min (mouse) and 8-42 min (human) (Table 6). The blood pharmacokinetics in
mice were measured following a single oral dose of 50 mg/kg. Peak blood levels (Cmax
and area under the curve (AUC) are shown in Table 6. Compound 2067 had the
highest Cmax (65 µM) and AUC (17263 min*µM/L), whereas compound 2093 had a
lower exposure in blood: Cmax (5.8 µM) and AUC (1863 min*µM/L). Levels of
compounds were also measured in the feces of selected compounds and were
observed to be in the 10-50 µM range with the exception of 2240 for which fecal levels
were very high at 846 µM.
)
Safety studies. The compounds with the best in vivo activity (2093, 2114, and 2259)
were tested for inhibition of five human CYP isoenzymes at a single 10 µM
concentration. Similar results were observed for all three compounds. Only CYP2C8
was inhibited by >50% at this concentration (Table 7). Inhibition of the hERG channels
was measured at two concentrations, 10 and 30 µM (Table 8). Again, similar results
were observed for three compounds. In the absence of serum, hERG inhibition was

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
approximately 60% at 30 µM, but the inhibition dropped to ~20% in the presence of
BSA. The Ames and in vitro micronucleus tests for genotoxicity were done on 2093 and
both found to be negative. Detailed results are available in the supplementary data
section.
DISCUSSION
The Cryptosporidium enzyme, methionyl-tRNA synthetase, was targeted for the
development of novel drugs to treat cryptosporidiosis. Analysis of the deposited amino
acid sequence of the CpMetRS revealed that it clusters with type 1 MetRS enzymes
(23) that include trypanosomes, Giardia, and Gram positive bacteria. In previous work,
crystal structures of the T. brucei MetRS revealed the ATP and methionine binding
pockets as well as the binding mode for numerous inhibitors (28, 29). By mapping the
C. parvum enzyme to the T. brucei MetRS structure, the analogous binding pocket was
identified, demonstrating that 19 of 25 amino acids (76%) forming the surface of the
pocket are identical. This indicated that inhibitors of the T. brucei MetRS would be likely
to bind the CpMetRS. The binding pocket residues of the C. hominis MetRS are
identical to those of C. parvum. The sequence analysis also showed that 18 of 24
residues (75%) were identical in the corresponding human mitochondrial MetRS. The
significance of this similarity is discussed below. In contrast, the human cytoplasmic
MetRS had a lower degree of identity, 14 of 24 (58%) which is consistent with the
knowledge that the human cytoplasmic MetRS belongs to the MetRS2 category which is
not inhibited by the compounds under investigation.
The CpMetRS gene was amplified from genomic DNA, cloned into an
expression vector, and overexpressed in E.coli. The purified enzyme was catalytically
3
active in an aminoacylation assay in which H-methionine is incorporated into tRNA
(Fig. S2, Supplementary Data). This method uses 100 nM of enzyme to provide an
acceptable signal to background ratio which constrains the ability to accurately measure
the K
enzyme concentration). In order to measure the K
exchange assay was adopted (31). In this method, incorporation of [ P]PP
occurs by the reverse enzyme reaction that can be measured when the receiving
substrate, tRNA, is omitted. The K for the enzyme substrates, methionine and ATP,
are within a factor of 2-3 of the values measured in this study and reported elsewhere
(31) for the S. aureus MetRS. In contrast, the K values of methionine and ATP for the
i
for highly potent inhibitors (since the IC50 can theoretically be no less than half the
i
for the MetRS inhibitors, the ATP:PP
i
3
2
i
into ATP
m
m
human mitochondrial MetRS are lower by a factor of 5-10.
Since the MetRS inhibitors are competitive with methionine (31), it is possible to
accurately measure the IC50s of inhibitors in the reaction by raising the concentration of
methionine above its K (34). The shift in IC50 is proportional to the [substrate]/K as
m m
described by the Cheng-Prusoff equation for competitive inhibitors (35). Of note, the
method of raising the methionine substrate concentration cannot be employed in the

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
aminoacylation assay mentioned earlier because the unlabeled methionine competes
3
with the H-methionine that is necessary for the readout. The K
i
values were
determined for selected MetRS inhibitors revealing extraordinary potency of many of
these compounds, ranging from 0.0009 to >1.33 nM (Table 4). This is similar to the K
for antibiotic MetRS inhibitor, REP8839 against the S. aureus MetRS, reported at 0.01
i
nM (31). The relationship between the K
i
and C. parvum EC50 showed a strong
2
correlation (R = 0.91; P value <0.0001) consistent with the observation that compound
2093 was the most potent compound against the MetRS enzyme and against
C. parvum infection of cell cultures. The correlation data support the conclusion that the
inhibitors act “on target” to mediate their effects on C. parvum cells.
More than 500 MetRS inhibitors have been developed in our program to optimize
their antitrypanosomal and antibacterial activities (24-26, 30). A set of structurally
diverse compounds from this library (Tables 3 and 4) was screened against C. parvum,
revealing a mix of positive and negative results. The aminoquinolone-containing
compounds (exemplified by 1312, (24)) had poor activity. The aminoquinolone-
compounds are in clinical development as antibiotics for Clostridium difficile and S.
aureus infections (36, 37). Similarly, compounds with the urea moiety (e.g. 1433, (25))
also had poor activity. In contrast, compounds with the fluoro-imidazopyridine (e.g.,
1614 and 1717, (26)) demonstrated more potent activity, in the 5-10 µM range. Parallel
work had indicated that changes to the linker region of the molecule were well tolerated,
leading to explorations of various changes including ring systems at this region of the
molecule (30). Compounds with the 1, 3-dihydro-2-oxo-imidazole as the linker ring were
particularly active (Table 4). Among the compounds containing the 1, 3-dihydro-2-oxo-
imidazole, various substitutions were explored at the R-groups indicated in Table 4. R
as ethyl (e.g., 2062) was more active than R as H (1962) or as propyl (2069). R1
1
as -CH
2
CF
2
H appears to be slightly less active than the ethyl version. R
2
as F is slightly
less active than R
2
as Cl; and Br is essentially the same as Cl. Next, substitutions on
the benzyl group (on the left-side of the structure, Table 4) were explored. It had been
previously shown that 3, 5-substitutions (such as 3 ,5 -dichlorobenzyl) were particularly
potent on the T. brucei MetRS (24). In the orientation created by the ring-linker
structures, the 2, 4-subsitutions (as shown in Table 4) have the greatest potency.
Changing R
4
from Cl to –OCH , produced the most potent inhibitors in the series (e.g.,
3
2093 and 2114). Compounds with a tri-substituted benzyl-group (e.g. 2, 4, 5-
substitutions) had much diminished activity against C. parvum oocysts (data not
shown). In summary, compound 2093 was the most potent compound (EC50 = 0.007
µM) and compares very favorably to the published data for the clinical drug,
nitazoxanide (EC50 = 3.7 µM), against C. parvum (38).
The activity of the MetRS inhibitors against C. parvum infection was tested in two
different murine models. The NOD SCID gamma mouse model was performed with a
PCR-based readout comparing pre-treatment (day 5) to post-treatment (day 11) fecal

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
parasite levels. Fecal oocysts were significantly reduced for 2093 (tested twice) as well
as for 2069 and 2259, but not for 2067. The activities were then retested in the
Cryptosporidium infection model using adult IFN- knockout mice. In this model, the
mice were monitored for 20 days post-infection. Quantitation of stool parasite loads
again showed that 2093 was highly efficacious (>4 log10 drop in luminescence) at the
oral dose of 50 mg/kg BID (Fig 3A). The greater magnitude of parasite reduction
observed in the IFN- knockout mice may be due to differences in the models and
readout methods. Additionally, the challenge dose was lower in the IFN- knockout
mice (10 ) compared to the NSG model (10 ) which may account for the differences if
there is an inoculum effect as seen with some bacterial infections (39). A persistence
of a low luminescence signal above background levels (log10 RLU of 2.5) was detected
during the remainder of the monitoring period in the IFN- model, the significance of
which is unclear. Importantly, the parasite signal did not rebound to the high levels
observed with the controls. Also, the mice maintained their body weight and survived to
the end of the 20 day observation period, unlike the vehicle-treated mice that needed to
be euthanized on day 17 due to loss of >20% body weight (Fig. 3A). Several follow up
experiments confirmed the in vivo activity of 2093 at lower doses (e.g. 50 mg/kg/day
divided in one or two doses), but the activity was substantially diminished at the dose of
20 mg/kg once per day. The other compounds with excellent in vivo activity were 2114
and 2259. The mice appeared to tolerate the treatments without any observed side
effects, and did not experience the weight loss that was observed in the vehicle-treated
mice. The one exception was for 2258 where the mice lost weight and were euthanized
on day 13 (before the control mice needed to be euthanized). For the other
3
5
compounds, it was encouraging to observe that potent anticryptosporidial activity was
associated with good clinical outcomes in the mice.
The compounds with the greatest in vivo efficacy (2093, 2114 and 2259) were
among the most potent in vitro compounds. To further understand the reasons for the
more potent in vivo activity, various physicochemical properties were assessed. Some
general characteristics of these potent in vivo compounds are molecular weights in the
400-450 range, log P in the 3.5-3.75 range, and relatively high solubility (>25 µM) at pH
levels reflective of the gastrointestinal tract and plasma. The MetRS inhibitors are all
highly protein-bound in the range of 95-99.9% (except for 2240 with 87.7% protein
binding). Apparently, high protein binding is not detrimental to activity since compound
2093 is 99.9% bound to mouse plasma proteins. Figure 4 allows for visualization of the
chemical properties as they relate to in vivo efficacy. It shows that the most active
compounds in vivo have relatively high solubility/EC50, apparently reflecting the
importance of in vitro potency (EC50) and, at least, moderate solubility. Interestingly, the
most effective compounds had intermediate levels of predicated permeability perhaps
suggesting that overly permeable compounds may be completely absorbed into the
blood and thus unavailable in the gut for local antiparasitic activity, and that compounds

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
with low permeability may not cross membranes sufficiently to exert effects on the
parasites either.
The liver microsome metabolic half-lives for the MetRS inhibitors were relatively
low (<15 min for most compounds tested, Table 6). In fact, the lead compound 2093
has a half-life of 3.2 and 8.3 minutes in murine and human microsomes, respectively.
This short in vitro half-life does not directly translate to low plasma exposure in vivo,
probably because of the protective effects of the high plasma protein binding.
The pharmacokinetic (PK) properties in mice of several of the MetRS inhibitors
were also assessed. The two most potent compounds (2093 and 2114) had similar PK
profiles with Cmax in the 6-8 µM range and AUC ~2000 min*umol/L. Fecal levels of 2093
and 2114 were 31 and 11 µM, respectively. The fecal levels suggest that sufficient
amounts of intact compound (>100 x EC50) are available in the fecal stream to exert
anti-cryptosporidiosis effects. Previous studies with “bumped kinase inhibitors” indicate
that intestinal levels of compound better correlate with anti-cryptosporidiosis activity
than do plasma levels (12). Both the fecal levels and plasma levels were similar
enough amongst the tested CpMetRS inhibitors thus it is not possible to make firm
conclusions about the most favorable properties. One exception is that compound 2240
had very high average fecal levels (846 µM) yet had no in vivo anti-Cryptosporidium
activity. We speculate that this compound may have passed through the GI tract in an
insoluble form that was not available for local anti-cryptosporidiosis activity. If this is true
then merely delivering insoluble compound to the gut is not sufficient. Since the
pathogen lives in an intracellular niche, it is necessary for the compound to be in
solution and sufficiently permeable to reach that niche either directly from the intestinal
lumen or via the bloodstream.
The safety of the compounds is of great importance given that the target
population for anti-cryptosporidiosis treatment will include very young children and other
vulnerable groups. A potential concern for MetRS inhibitors is cross-activity on the
human mitochondrial MetRS enzyme that could lead to mitochondrial dysfunction. The
human mitochondrial MetRS and the CpMetRS are identical at 18 of 25 residues in the
compound binding pocket (see Table 1), indicating moderate similarity. An assay was
performed to quantify cytochrome oxidase 1 (COX-1) enzyme levels in human liver cells
(HepG2) after six-day incubation with MetRS inhibitors. This enzyme is encoded and
expressed in the mitochondrion, whereas the control protein (SDH-A) is encoded in the
nucleus and expressed in the cytoplasm. The single ring-linker MetRS inhibitors (Table
4), in fact, demonstrated substantial inhibition of COX-1 expression levels with EC50
values as low as 0.039 µM in the case of 2093. This is only slightly above the EC50
value against C. parvum (0.007 – 0.036 µM). The other single ring-linker compounds
generally had EC50 values <0.5 µM in this assay. The in vivo effects that may result
from mitochondrial protein synthesis inhibition will require further investigation. It is
worth noting that many antibiotics that work by inhibiting prokaryotic protein synthesis

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
also inhibit mitochondrial protein synthesis (40). For example, the commonly used
antibiotics such as doxycycline (COX-1 EC50 = 6.6 µM) and linezolid (COX-1 EC50 = 15
µM) are used at plasma levels that approximate or exceed the EC50 concentrations in
the COX-1 assay (41, 42). In the case of linezolid, toxicity due to mitochondrial inhibition
can be observed during normal use of the drug, although this typically does not become
serious until after four weeks of treatment (43). With anticipated treatment courses for
cryptosporidiosis being relatively short (ideally no more than three days), it is likely that
brief exposures to MetRS inhibitors would be well tolerated, although clearly this will
require careful investigation. If mitochondrial inhibition is a problem, then additional
effort to identify more selective compounds will be pursued in future work.
The compounds with the best in vivo activity (2093, 2114, and 2259) were also
tested for inhibition of the hERG channels and CYP450 enzymes. The hERG inhibition
assay screens for potential of the compound to promote dangerous cardiac
dysrhythmias. In the presence of plasma protein (BSA), the percent inhibition was only
~20% at concentrations of 30 µM which is reassuring. The concentration that
substantially inhibits hERG channels is likely to be several multiples above the peak
concentrations that would occur during treatment. This same compound had little effect
on CYP450 enzymes except for CYP2C8 (causing 62-87% inhibition at 10 µM).
Common drugs that are metabolized by CYP2C8 include: rosiglitazone (antidiabetic),
montelukast (asthma), cerivastatin (statin), and amodiaquine (antimalarial). The co-
administration of these MetRS inhibitors with the listed drugs could potentially lead to
drug-drug interactions. Finally, Ames and in vitro micronucleus tests were done on
compound 2093 and were negative. This provides reassurance that 2093 is not
genotoxic.
In summary, the imidazopyridine compounds described herein have potent
activity against the C. parvum MetRS enzyme as well as against cultures of C. parvum
and C. hominis. Parasite persistence assays suggest the compounds have parasiticidal
effects on the parasites. Most importantly, the MetRS inhibitors controlled C. parvum
infection in two murine models without producing side effects. The active compounds
demonstrated substantial plasma exposures as well as fecal levels, but it is not entirely
clear from these data which of these pharmacological parameters is most relevant. The
MetRS inhibitors are capable of inhibiting the human mitochondrial MetRS enzyme as
determined by reduced levels of a mitochondrial protein (COX-1) in cultured HepG2
cells; however, clinical toxicity may be unlikely if the duration of treatment is kept to
short durations (e.g. <1 week). Future studies with 2093 and other MetRS inhibitors in
the calf Cryptosporidium infection model as well as in vivo toxicology studies will help
establish the potential for developing these compounds for treatment of human
cryptosporidiosis.
4
59
METHODS

4
4
60
61
Protein sequence alignments: Global pairwise amino acid sequence alignments were
generated with the NCBI alignment tool Clustal Omega (44).
4
4
4
4
4
4
62
63
64
65
66
67
CpMetRS cloning, expression, and purification: The CpMetRS gene (UniProtKB
accession number Q5CVN0) was PCR amplified from genomic DNA isolated from the
C. parvum Iowa II strain. The PCR product was then cloned into the AVA0421 plasmid
(45), and the sequence was verified. The expression of the recombinant protein was
performed as previously described (45). The protein was purified by nickel affinity
chromatography followed by size-exclusion chromatography.
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
Enzyme assays: Inhibition of CpMetRS was measured using an ATP:PP exchange
assay as previously described (31, 46) with some modifications. The compounds were
i
pre-incubated for 5 min at room temperature in a 96 well-plate with 30 nM CpMetRS,
125 mM L-methionine, 25 µM NaPP
, ~2 µCi of 32P-tetrasodium pyrophosphate
i
(NEX019001MC, PerkinElmer), 2.5 mM dithiothreitol, 100 mM Tris-HCl pH 8.0, 10 mM
magnesium acetate, 80 mM KCl, and 2% dimethyl sulfoxide (DMSO). The reaction was
started with the addition of 25 µM ATP, after a 10 min incubation at room temperature, 5
µL of the reaction (in duplicate) was quenched into a MultiScreenHTS DuraporeTM 96-well
filter plate (MSHVN4B50, Millipore Sigma) containing a mixture of 200 µL of 10%
charcoal with 0.5% HCl and 50 µL of 1M HCl with 200 mM of sodium pyrophosphate.
The filter plates were washed three times with 200 µL of 1M HCl with 200 mM of sodium
pyrophosphate on a vacuum manifold. The plates were dried for 30 minutes at room
temperature and then 25 µL of scintillation cocktail was added. Plates were incubated
at room temperature for ~1.5 hours before counts per minute (CPM) were quantified on
the MicroBeta2 scintillation counter (PerkinElmer). Percent inhibition was calculated by
subtracting off the background wells (containing all assay reagents except ATP and
compound) and comparing this to the high control wells (containing all assay reagents
without compound). IC50s were calculated by non-linear regression methods using the
Collaborative Drug Database (Burlingame, CA. www.collaborativedrug.com). K
calculated from the IC50s that were shifted above the enzyme concentration (30 nM)
using the Cheng-Prusoff equation: IC50 = (1+[Met]/K (31). The
Met)(1+K ATP/[ATP)K
s for L-methionine were determined using the same assay conditions above with
either SaMetRS or CpMetRS (30 nM) without compounds and with ~5 µCi of 32P-
tetrasodium pyrophosphate, 2.5 mM of ATP, and 2.5 mM of NaPP while titrating the L-
methionine. The reaction was quenched as described above at different time intervals,
typically 0, 4, 8, 12, 16, and 20 min. Similarly, the K s for ATP were measured by using
1 mM of L-methionine and 2.5 mM of NaPP while titrating the ATP. K and Vmax values
i
s were
m
m
i
K
m
i
m
i
m
were calculated in Prism (version 3.0) software. Kcat is equal to the Vmax divided by the
enzyme concentration (30 nM).

4
4
5
5
98
99
00
01
Chemistry: Compound synthesis for 1312 (24),1433 (25), 1614 (26), 1717 (26), 1962
(27), 2062 (27), 2093 (27), and 2114 (27) were reported previously. Synthesis of
compounds 2067, 2069, 2080, 2091, 2138, 2139, 2207, 2240, 2242, 2258, and 2259
are described in the supplemental material.
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Propogation methods and growth inhibition assays for C. parvum: Genetically
modified Nanoluciferase (Nluc) expressing C. parvum Iowa strain oocysts were
propagated in female interferon-γ knockout mice (C57BL/6 IFN-c-deficient mice
B6.129S7-Ifngtm1Ts/J, The Jackson Laboratory, Bar Harbor, ME) (47). Mice were
infected by oral gavage with 1,000 oocysts in 0.1 mL DPBS (Sigma, St. Louis, MO).
Fecal samples were collected starting 3-5 days after infection. Multiple times per week
for 2 to 3 weeks, mice were transferred to a clean cage for 1-2 hours, and feces were
collected and stored in a 2.5% potassium dichromate solution at 4°C. Oocysts were
purified from feces using sucrose flotation followed by cesium chloride gradient as
previously described (48).
Growth inhibition assays at University of Washington were performed as follows.
HCT-8 cells were added to a 96-well plate and allowed to grow for 72 h to reach 90-
100% confluence. Then the media was removed and test compounds were added in
serial dilutions prior to the addition of 1,000 oocysts per well in 0.1 mL RPMI-1640
medium supplemented with 10% horse serum and 1% penicillin/streptomycin. Plates
were incubated for 72 h and then Nano-Glo® luciferase reagent (Promega, Madison,
WI) was added and plates were read on an EnVision Multilabel Plate Reader (Perkin
Elmer, Waltham, MA, USA). EC50 curves were calculated as previously described using
GraphPad Prism version 6.07 (GraphPad Software, La Jolla, CA) (33).
C. parvum growth inhibition assays completed at the University of Vermont were
performed as described previously using wild-type C. parvum Iowa strain oocysts
freshly isolated from calves (purchased from Bunch Grass Farm, Deary, ID) and high
content microscopy (32, 38). Excystation of oocysts was induced by treatment with 10
mM hydrochloric acid (10 min at 37°C), followed by exposure to 2 mM sodium
taurocholate (Sigma-Aldrich) in PBS for 10 min at 16°C. Excysted oocysts were then
added to 95% confluent HCT-8 cell monolayers in 384-well plates (~5,500 oocysts per
well). Compounds were added 3 h after infection, and assay plates were incubated for
48 h post-infection at 37°C under 5% CO2. The cell monolayers were then washed
three times with PBS containing 111 mM -galactose, fixed with 4% paraformaldehyde,
D
permeabilized with 0.25% Triton X-100, and blocked overnight with 4% bovine serum
albumin (BSA) in PBS. Parasitophorous vacuoles were stained with 1.33 µg/ml of
fluorescein-labeled Vicia villosa lectin (Vector Laboratories) diluted in 1% BSA in PBS
with 0.1% Tween 20 for 1 h at 37°C, followed by the addition of Hoechst 33258
(AnaSpec) at a final concentration of 0.09 mM diluted in water for another 15 min at
37°C. Wells were then washed five times with PBS containing 0.1% Tween 20. A Nikon
Eclipse TE2000 epifluorescence microscope with an automated stage was programmed

5
5
5
5
5
5
5
5
38
39
40
41
42
43
44
45
to focus on the center of each well and take a 3-by-3 composite image using an EXi
Blue fluorescence microscopy camera (QImaging, Canada) with a 20X objective
(numerical aperture, 0.45). Nucleus and parasite images were exported separately as
tiff files and were analyzed on the ImageJ platform (National Institutes of Health) using
previously developed macros (38).
The methods for screening C. hominis and clinical isolates of C. parvum from
dairy calves (reported in supplementary data) were the same as reported previously
(49). The strains were collected by Dr. McNamara at CALIBR (32).
5
5
5
5
5
5
5
5
46
47
48
49
50
51
52
53
Parasite persistence assays: The procedures were previously published (32). Briefly,
HCT-8 cell monolayer infections were established as above in 384-well culture plates
using wild-type C. parvum Iowa strain oocysts. After approximately 24 h, compounds
were added at various concentrations as labeled, and then parasites and host cells
were enumerated at multiple time points using immunofluorescence microscopy. Data
points are means and standard deviations for 4 culture wells per time point and are
representative of 3 independent experiments. The P value is the replicates test result
(note that a P value of ≥0.05 indicates a valid curve fit).
5
5
5
5
5
5
5
54
55
56
57
58
59
60
Mammalian cell growth inhibition assays: Compounds were tested against CRL-
8155, human lymphocytic cells, and HepG2, human hepatocellular cells, as previously
described (50). Briefly, compounds were incubated with CRL-8155 cells (30,000/well)
or HepG2 cells (25,000/well) for 48 hours in 96-well plates and then developed using
AlamarBlue® (ThermoFisher Scientific). The EC50 values were calculated from percent
inhibition by non-linear regression methods using the Collaborative Drug Discovery
database (Burlingame, CA. www.collaborativedrug.com) as previously described (50).
5
5
5
5
5
5
5
5
5
5
5
61
62
63
64
65
66
67
68
69
70
71
MitoBiogenesisTM In-Cell ELISA Colorimetric assay: 24 hours before adding
compounds, 6,000 HepG2 (human hepatocellular) cells were seeded per well into
GibcoTM Collagen I, Coated Plate, 96 Well (A1142803,ThermoFisher Scientific) in
culture media (50). The next day, the media was removed and compounds were added
in culture media. Every 48 hours thereafter during the 6 day incubation period, the
media with compounds was replaced with freshly diluted compounds in media. Plates
TM
were then fixed with 4% paraformaldehyde and developed using the MitoBiogenesis
In-Cell ELISA Kit (Colorimetric) (ab110217, Abcam) according to the manufacturer’s
instructions. The EC50 values were calculated from the normalized data by non-linear
regression methods using the Collaborative Drug Discovery database (Burlingame, CA.
www.collaborativedrug.com) or in Prism (version 3.0) software.
5
5
5
5
72
73
74
75
Mouse efficacy models: The efficacy in adult interferon γ (IFN-γ) knockout (KO) mice
completed at the University of Washington was carried out as previously described (33)
and were approved by the University of Washington Institutional Animal Care and Use
Committee. Briefly, female IFN-γ KO mice (B6.129S7-Ifngtm1Ts/J, Jackson

5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
Laboratories) aged 8–10 weeks were infected by oral gavage with 1,000
Nanoluciferase-tagged C. parvum UGA1 oocysts in 0.1 mL of Dulbecco’s phosphate-
buffered saline. Each experimental group included 3 mice. On day 6 post-infection,
mice were administered compounds orally in a formulation of 3% ethanol/7% Tween
80/90% saline for 5 days. The fecal samples were collected out to day 20 post infection
and luminescences was quantified in Relative Light Units and normalized as previously
described (33).
All NOD SCID gamma mouse studies were performed in compliance with animal
care guidelines and were approved by the University of Vermont Institutional Animal
Care and Use Committee. Male NOD SCID gamma mice with normal flora (NOD.Cg-
Prkdcscid Il2rgtm1Wjl/SzJ) (51) were purchased from The Jackson Laboratory (Bar Harbor,
ME, USA) and were housed for at least a week for acclimatization. At the age of 4 to 5
5
weeks, mice were infected by oral gavage with 10 C. parvum Iowa strain oocysts.
Treatment was started on day 6 after infection. Mice (4 per experimental group) were
treated orally (p.o.) with test compounds at the indicated doses from days 6-10 post-
infection. Oocyst shedding in feces was monitored using a previously validated
quantitative PCR (qPCR) assay and primers (52).
5
5
5
5
5
5
5
6
6
6
6
6
6
93
94
95
96
97
98
99
00
01
02
03
04
05
Measurements of compound solubility: Two microliters of 20 mM dimethyl sulfoxide
stock solution of compound were added to 398 µL of pH = 7.4, 2.0, or 6.5 in PBS buffer.
The mixture was vigorously shaken to mix the sample thoroughly, and then the sample
was incubated at 25 C overnight. The mixture was centrifuged at 25 C for 20 min at
15,000xg. The supernatant 100 µL was transferred into another vial and diluted with 100
◦
◦
µL CH
system.
3
CN. The two-fold diluted supernatant (100 µL) was analyzed by HPLC/ UV
Measurements of plasma protein binding: Compound binding to mouse plasma
proteins was determined using Rapid Equilibrium Dialysis device (catalog number
89809; ThermoFisher Scientific) or 96-well equilibrium dialyzer plates (catalog number
SDIS 9610EN; Nest Group, Inc.) or in-house made micro dialysis plates (53) according
to published methods (27, 53) and manufacture’s instruction (54).
6
6
6
6
6
6
6
06
07
08
09
10
11
12
Parallel artificial membrane permeability assay (PAMPA): The donor well was filled
with 200 μL of PRISMA HT buffer (pH 5.0 or pH 7.4, pION inc.) containing 10 μM test
compound. The filter on the bottom of each acceptor well was coated with 4 μL of a
GIT-0 Lipid Solution (pION, Inc.) and filled with 200 μL of Acceptor Sink Buffer (pION,
Inc.). The acceptor filter plate was put on the donor plate and incubated for 3 hours.
After the incubation, the amount of test compound in both the donor and acceptor wells
was measured by LC-MS/MS to calculate the permeability rate.
6
6
6
13
14
15
Microsome stability assays: Liver microsomes were purchased from Sekisui
XenoTech, LLC (Kansas City, KS, USA). The microsomes (0.2 mg protein/mL) and the
compound (1 µM) were mixed in phosphate buffer (pH 7.4). The reactions were initiated

6
6
6
6
6
6
6
6
6
6
16
17
18
19
20
21
22
23
24
25
by adding an NADPH generating system (a mixture of MgCl2, β-NADP+, glucose-6-
phosphate, and glucose-6-phosphate dehydrogenase) to the mixtures before
incubation. Incubations were conducted at 37ºC and terminated by adding acetonitrile.
The zero-time incubations, which served as the controls, were terminated by adding
acetonitrile before adding an NADPH generating system. After the samples were mixed
and centrifuged, the compound concentration in the supernatant fractions were
measured by LC/MS/MS. Control compounds demonstrated the following in vitro
clearance rates: flutamide: 161 µL/min/mg (human) and 304 µL/min/mg (mouse);
quinidine: 4 µL/min/mg (human) and 27 µL/min/mg (mouse); and cilostazol: 28
µL/min/mg (human) and 100 µL/min/mg (mouse);
6
26
6
6
6
6
6
6
6
6
6
6
6
6
27
28
29
30
31
32
33
34
35
36
37
38
CYP450 Inhibition assays: Human liver microsomes were purchased from Sekisui
XenoTech, LLC (Kansas City, KS, USA). The microsomes (0.1 mg protein/mL),
substrates (tacrine, paclitaxel, tolbutamide, dextromethorphan, and midazolam) and the
compound (10 µM) were mixed in phosphate buffer (pH 7.4). The reactions were
initiated by adding an NADPH generating system (a mixture of MgCl2, β-NADP+,
glucose-6-phosphate, and glucose-6-phosphate dehydrogenase) to the mixtures before
incubation. Incubations were conducted at 37°C for 10 minutes and terminated by
adding acetonitrile. The activities of CYP1A2, CYP2C8, CYP2C9, CYP2D6 and
CYP3A4 were determined by the peak of 1-hydroxytacrine, 6α-hydroxypaclitaxel, 4-
hydroxytolbutamide, dextrorphan and 1'-hydroxymidazolam, respectively. The activities
of test samples were expressed as the percentage of activity remaining compared with
a control sample containing no inhibitor.
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
hERG inhibition assays: hERG/CHO cells stably expressing hERG channel were
purchased from Millipore, Ltd (UK). Cells were cultured at 32 ºC, 5% CO2 in Ham’s F-
12 medium supplemented with 10% fetal bovine serum, 500ug/mL Geneticin . The
hERG inhibition assay was performed on the IonWorks Quattro (Molecular Devices)
system in population patch clamp (PPC) mode. The extracellular solution was
phosphate-buffered saline with calcium and magnesium. The intracellular solution
contained 120 µM amphotericin B, 140 mM KCl, 2 mM MgCl2, 1 mM EGTA and 20 mM
HEPES (pH 7.3). The hERG current was measured under the potential-clamp protocol
(Holding potential -80 mV, the first voltage 40 mV for 2 sec, the second voltage -50 mV
for 2 sec). After patch perforation, the peak tail current before addition of the
compounds were measured as the pre-hERG current. Test compounds were incubated
on the cells for a period of 5 min. The peak tail current after addition of the
compounds were measured as the post-hERG current. The hERG inhibition assays
were performed in triplicate in two kinds of extracellular solutions which contained 1%
BSA or none.

6
6
6
6
6
6
6
6
54
55
56
57
58
59
60
61
Mouse pharmacokinetics: Nonfasted female Swiss Webster mice (n=3) were
administered 50 mpk of compound by oral gavage in a vehicle consisting of 7% Tween
80, 3% ethanol, 5% DMSO, and 0.9% saline. Tail blood was collected at 30, 60, 120,
240, 360, 480, and 1,440 min into heparinized capillary tubes and spotted onto
Whatman Gel Blotting paper as previously described (50). Whole-blood samples were
extracted with acetonitrile and analyzed by liquid chromatography-tandem mass
spectrometry. The values of the pharmacokinetic parameters were calculated using
Phoenix WinNonlin (version 6.3) software (Certara, Princeton, NJ).
6
6
6
6
6
6
6
6
6
6
6
6
6
6
62
63
64
65
66
67
68
69
70
71
72
73
74
75
Feces were collected and pooled from all three mice from the entire duration of the
pharmacokinetic experiment described above (0 min to 1,440 min). The pellets were
suspended in 4x water by weight and homogenized. To extract compounds of interest,
acetonitrile (8x by volume) was added to the homogenate containing 100 mg of feces
followed by addition of internal standard solution (90% acetonitrile : 10% water). After
centrifuging the homogenate solution, the supernatant was dried in a speed-vac. The
dried samples were reconstituted in a LC-MS sample solution (50% MQ water : 50%
Acetonitrile). The solution was centrifuged and the supernatant was transferred to a
liquid chromatography insert. Each compound was tested in triplicate. Similarly, fecal
homogenate from vehicle mice were prepared to make calibration standards. The
compound concentrations for calibration standards were 0 nM, 10 nM, 100 nM, 1 µM, 5
µM, 10 µM, 20 µM, 40 µM. The compound concentrations for each homogenate from
treated mice group were calculated from the calibration curves using Microsoft Excel
software.
6
76

6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
ACKNOWLEDGEMENTS: Support for this project was provided by PATH with funding
from the UK government, and by the National Institutes of Health (grant AI097177). The
purified recombinant C. parvum MetRS protein (UniProt Q5CVN0) described herein was
provided by the Seattle Structural Genomics Center for Infectious Disease
(www.SSGCID.org) supported by Federal Contract No. HHSN272201700059C from the
National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Department of Health and Human Services. We acknowledge the contributions of the
following individuals: Nora Molasky, Uyen Nguyen, and Omeed Faghih for performing
mammalian cell cytotoxicity assays; Molly McCloskey and Grant Whitman for assisting
with in vivo efficacy experiments; Zack Herbst for analytical chemistry on
pharmacokinetic assays; and Hannah Udell for cloning and purifying the C. parvum
MetRS enzyme. We acknowledge contributions of project oversight by Eugenio de
Hostos (PATH). We also acknowledge valuable contributions by collaborating scientists
at Takeda (Yasushi Miyazaki) and CALIBR (Melissa Love and Case McNamara).
6
92
6
93
REFERENCES
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
1.
2.
3.
Nations U. https://www.un.org/sustainabledevelopment/sustainable-development-goals/.
Accessed
Organization WH. 2018. Global Health Estimates 2016: Deaths by Cause, Age, Sex, by Country
and by Region, 2000-2016. . World Health Organization, Geneva.
Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D,
Breiman RF, Faruque AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U,
Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK,
Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ, Akinsola A, Mandomando I,
Nhampossa T, Acacio S, Biswas K, O'Reilly CE, Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H,
Robins-Browne RM, Levine MM. 2013. Burden and aetiology of diarrhoeal disease in infants and
young children in developing countries (the Global Enteric Multicenter Study, GEMS): a
prospective, case-control study. Lancet 382:209-22.
Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, Haque R, Havt A, McCormick BJ, McGrath M,
Olortegui MP, Samie A, Shakoor S, Mondal D, Lima IF, Hariraju D, Rayamajhi BB, Qureshi S, Kabir
F, Yori PP, Mufamadi B, Amour C, Carreon JD, Richard SA, Lang D, Bessong P, Mduma E, Ahmed
T, Lima AA, Mason CJ, Zaidi AK, Bhutta ZA, Kosek M, Guerrant RL, Gottlieb M, Miller M, Kang G,
Houpt ER, Investigators M-EN. 2015. Pathogen-specific burdens of community diarrhoea in
developing countries: a multisite birth cohort study (MAL-ED). Lancet Glob Health 3:e564-75.
Korpe PS, Haque R, Gilchrist C, Valencia C, Niu F, Lu M, Ma JZ, Petri SE, Reichman D, Kabir M,
Duggal P, Petri WA, Jr. 2016. Natural History of Cryptosporidiosis in a Longitudinal Study of
Slum-Dwelling Bangladeshi Children: Association with Severe Malnutrition. PLoS Negl Trop Dis
10:e0004564.
4.
5.
6.
Khalil IA, Troeger C, Rao PC, Blacker BF, Brown A, Brewer TG, Colombara DV, De Hostos EL,
Engmann C, Guerrant RL, Haque R, Houpt ER, Kang G, Korpe PS, Kotloff KL, Lima AAM, Petri WA,
Jr., Platts-Mills JA, Shoultz DA, Forouzanfar MH, Hay SI, Reiner RC, Jr., Mokdad AH. 2018.
Morbidity, mortality, and long-term consequences associated with diarrhoea from

7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
Cryptosporidium infection in children younger than 5 years: a meta-analyses study. Lancet Glob
Health 6:e758-e768.
7.
Checkley W, White AC, Jr., Jaganath D, Arrowood MJ, Chalmers RM, Chen XM, Fayer R, Griffiths
JK, Guerrant RL, Hedstrom L, Huston CD, Kotloff KL, Kang G, Mead JR, Miller M, Petri WA, Jr.,
Priest JW, Roos DS, Striepen B, Thompson RC, Ward HD, Van Voorhis WA, Xiao L, Zhu G, Houpt
ER. 2015. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for
cryptosporidium. Lancet Infect Dis 15:85-94.
Clode PL, Koh WH, Thompson RCA. 2015. Life without a Host Cell: What is Cryptosporidium?
Trends Parasitol 31:614-624.
Shrivastava AK, Kumar S, Smith WA, Sahu PS. 2017. Revisiting the global problem of
cryptosporidiosis and recommendations. Trop Parasitol 7:8-17.
Mor SM, Tumwine JK, Ndeezi G, Srinivasan MG, Kaddu-Mulindwa DH, Tzipori S, Griffiths JK.
2010. Respiratory cryptosporidiosis in HIV-seronegative children in Uganda: potential for
respiratory transmission. Clin Infect Dis 50:1366-72.
Mor SM, Ascolillo LR, Nakato R, Ndeezi G, Tumwine JK, Okwera A, Sponseller JK, Tzipori S,
Griffiths JK. 2018. Expectoration of Cryptosporidium Parasites in Sputum of Human
Immunodeficiency Virus-Positive and -Negative Adults. Am J Trop Med Hyg 98:1086-1090.
Arnold SLM, Choi R, Hulverson MA, Schaefer DA, Vinayak S, Vidadala RSR, McCloskey MC,
Whitman GR, Huang W, Barrett LK, Ojo KK, Fan E, Maly DJ, Riggs MW, Striepen B, Van Voorhis
WC. 2017. Necessity of Bumped Kinase Inhibitor Gastrointestinal Exposure in Treating
Cryptosporidium Infection. J Infect Dis 216:55-63.
Nakama T, Nureki O, Yokoyama S. 2001. Structural basis for the recognition of isoleucyl-
adenylate and an antibiotic, mupirocin, by isoleucyl-tRNA synthetase. J Biol Chem 276:47387-
47393.
Rock FL, Mao W, Yaremchuk A, Tukalo M, Crepin T, Zhou H, Zhang YK, Hernandez V, Akama T,
Baker SJ, Plattner JJ, Shapiro L, Martinis SA, Benkovic SJ, Cusack S, Alley MR. 2007. An antifungal
agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site. Science
316:1759-1761.
Elewski BE, Aly R, Baldwin SL, Gonzalez Soto RF, Rich P, Weisfeld M, Wiltz H, Zane LT, Pollak R.
2015. Efficacy and safety of tavaborole topical solution, 5%, a novel boron-based antifungal
agent, for the treatment of toenail onychomycosis: Results from 2 randomized phase-III studies.
J Am Acad Dermatol 73:62-9.
Keller TL, Zocco D, Sundrud MS, Hendrick M, Edenius M, Yum J, Kim YJ, Lee HK, Cortese JF, Wirth
DF, Dignam JD, Rao A, Yeo CY, Mazitschek R, Whitman M. 2012. Halofuginone and other
febrifugine derivatives inhibit prolyl-tRNA synthetase. Nat Chem Biol 8:311-317.
O'Dwyer K, Spivak AT, Ingraham K, Min S, Holmes DJ, Jakielaszek C, Rittenhouse S, Kwan AL, Livi
GP, Sathe G, Thomas E, Van HS, Miller LA, Twynholm M, Tomayko J, Dalessandro M, Caltabiano
M, Scangarella-Oman NE, Brown JR. 2015. Bacterial resistance to leucyl-tRNA synthetase
inhibitor GSK2251052 develops during treatment of complicated urinary tract infections.
Antimicrob Agents Chemother 59:289-298.
Hernandez V, Crepin T, Palencia A, Cusack S, Akama T, Baker SJ, Bu W, Feng L, Freund YR, Liu L,
Meewan M, Mohan M, Mao W, Rock FL, Sexton H, Sheoran A, Zhang Y, Zhang YK, Zhou Y,
Nieman JA, Anugula MR, Keramane el M, Savariraj K, Reddy DS, Sharma R, Subedi R, Singh R,
O'Leary A, Simon NL, De Marsh PL, Mushtaq S, Warner M, Livermore DM, Alley MR, Plattner JJ.
2013. Discovery of a novel class of boron-based antibacterials with activity against gram-
negative bacteria. Antimicrob Agents Chemother 57:1394-403.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Li X, Hernandez V, Rock FL, Choi W, Mak YSL, Mohan M, Mao W, Zhou Y, Easom EE, Plattner JJ,
Zou W, Perez-Herran E, Giordano I, Mendoza-Losana A, Alemparte C, Rullas J, Angulo-Barturen I,

7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
Crouch S, Ortega F, Barros D, Alley MRK. 2017. Discovery of a Potent and Specific M.
tuberculosis Leucyl-tRNA Synthetase Inhibitor: (S)-3-(Aminomethyl)-4-chloro-7-(2-
hydroxyethoxy)benzo[c][1,2]oxaborol-1(3H)-ol (GSK656). J Med Chem 60:8011-8026.
Palencia A, Li X, Bu W, Choi W, Ding CZ, Easom EE, Feng L, Hernandez V, Houston P, Liu L,
Meewan M, Mohan M, Rock FL, Sexton H, Zhang S, Zhou Y, Wan B, Wang Y, Franzblau SG,
Woolhiser L, Gruppo V, Lenaerts AJ, O'Malley T, Parish T, Cooper CB, Waters MG, Ma Z, Ioerger
TR, Sacchettini JC, Rullas J, Angulo-Barturen I, Perez-Herran E, Mendoza A, Barros D, Cusack S,
Plattner JJ, Alley MR. 2016. Discovery of Novel Oral Protein Synthesis Inhibitors of
Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase. Antimicrob Agents Chemother
60:6271-80.
Pham JS, Dawson KL, Jackson KE, Lim EE, Pasaje CF, Turner KE, Ralph SA. 2014. Aminoacyl-tRNA
synthetases as drug targets in eukaryotic parasites. Int J Parasitol Drugs Drug Resist 4:1-13.
Palencia A, Liu RJ, Lukarska M, Gut J, Bougdour A, Touquet B, Wang ED, Li X, Alley MR, Freund
YR, Rosenthal PJ, Hakimi MA, Cusack S. 2016. Cryptosporidium and Toxoplasma Parasites Are
Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase. Antimicrob Agents Chemother
60:5817-27.
Gentry DR, Ingraham KA, Stanhope MJ, Rittenhouse S, Jarvest RL, O'Hanlon PJ, Brown JR,
Holmes DJ. 2003. Variable sensitivity to bacterial methionyl-tRNA synthetase inhibitors reveals
subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase
genes. Antimicrob Agents Chemother 47:1784-1789.
Shibata S, Gillespie JR, Kelley AM, Napuli AJ, Zhang Z, Kovzun KV, Pefley RM, Lam J, Zucker FH,
Van Voorhis WC, Merritt EA, Hol WG, Verlinde CL, Fan E, Buckner FS. 2011. Selective Inhibitors
of Methionyl-tRNA Synthetase Have Potent Activity against Trypanosoma brucei Infection in
Mice. Antimicrob Agents Chemother 55:1982-1989.
Shibata S, Gillespie JR, Ranade RM, Koh CY, Kim JE, Laydbak JU, Zucker FH, Hol WG, Verlinde CL,
Buckner FS, Fan E. 2012. Urea-based inhibitors of Trypanosoma brucei methionyl-tRNA
synthetase: selectivity and in vivo characterization. J Med Chem 55:6342-6351.
Zhang Z, Koh CY, Ranade RM, Shibata S, Gillespie JR, Hulverson MA, Nguyen J, Pendem N, Gelb
MH, Verlinde CL, Hol WG, Buckner FS, Fan E. 2016. 5-Fluoro-imidazo[4,5-b]pyridine is a
privileged fragment that conveys bioavailability to potent trypanosomal methionyl-tRNA
synthetase inhibitors. ACS Infect Dis 2:399-404.
Faghih O, Zhang Z, Ranade RM, Gillespie JR, Creason SA, Huang W, Shibata S, Barros-Alvarez X,
Verlinde C, Hol WGJ, Fan E, Buckner FS. 2017. Development of Methionyl-tRNA Synthetase
Inhibitors as Antibiotics for Gram-Positive Bacterial Infections. Antimicrob Agents Chemother
61.
Koh CY, Kim JE, Shibata S, Ranade RM, Yu M, Liu J, Gillespie JR, Buckner FS, Verlinde CL, Fan E,
Hol WG. 2012. Distinct States of Methionyl-tRNA Synthetase Indicate Inhibitor Binding by
Conformational Selection. Structure 20:1681-1691.
Koh CY, Kim JE, Wetzel AB, WJ dvdS, Shibata S, Ranade RM, Liu J, Zhang Z, Gillespie JR, Buckner
FS, Verlinde CL, Fan E, Hol WG. 2014. Structures of Trypanosoma brucei methionyl-tRNA
synthetase with urea-based inhibitors provide guidance for drug design against sleeping
sickness. PLoS Negl Trop Dis 8:e2775.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
Huang W, Zhang Z, Barros-Alvarez X, Koh CY, Ranade RM, Gillespie JR, Creason SA, Shibata S,
Verlinde CL, Hol WG, Buckner FS, Fan E. 2016. Structure-guided design of novel Trypanosoma
brucei Methionyl-tRNA synthetase inhibitors. Eur J Med Chem 124:1081-1092.
Green LS, Bullard JM, Ribble W, Dean F, Ayers DF, Ochsner UA, Janjic N, Jarvis TC. 2009.
Inhibition of methionyl-tRNA synthetase by REP8839 and effects of resistance mutations on
enzyme activity. Antimicrob Agents Chemother 53:86-94.

8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
32.
33.
34.
35.
Jumani RS, Bessoff K, Love MS, Miller P, Stebbins EE, Teixeira JE, Campbell MA, Meyers MJ,
Zambriski JA, Nunez V, Woods AK, McNamara CW, Huston CD. 2018. A Novel Piperazine-Based
Drug Lead for Cryptosporidiosis from the Medicines for Malaria Venture Open-Access Malaria
Box. Antimicrob Agents Chemother 62.
Hulverson MA, Vinayak S, Choi R, Schaefer DA, Castellanos-Gonzalez A, Vidadala RSR, Brooks CF,
Herbert GT, Betzer DP, Whitman GR, Sparks HN, Arnold SLM, Rivas KL, Barrett LK, White AC, Jr.,
Maly DJ, Riggs MW, Striepen B, Van Voorhis WC, Ojo KK. 2017. Bumped-Kinase Inhibitors for
Cryptosporidiosis Therapy. J Infect Dis 215:1275-1284.
Pope AJ, Moore KJ, McVey M, Mensah L, Benson N, Osbourne N, Broom N, Brown MJ, O'Hanlon
P. 1998. Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. II.
Mechanism of inhibition by reaction intermediate and pseudomonic acid analogues studied
using transient and steady-state kinetics. J Biol Chem 273:31691-701.
Cheng Y, Prusoff WH. 1973. Relationship between the inhibition constant (K1) and the
concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.
Biochem Pharmacol 22:3099-108.
Critchley IA, Ochsner UA. 2008. Recent advances in the preclinical evaluation of the topical
antibacterial agent REP8839. Curr Opin Chem Biol 12:409-417.
Critchley IA, Green LS, Young CL, Bullard JM, Evans RJ, Price M, Jarvis TC, Guiles JW, Janjic N,
Ochsner UA. 2009. Spectrum of activity and mode of action of REP3123, a new antibiotic to treat
Clostridium difficile infections. J Antimicrob Chemother 63:954-963.
Bessoff K, Sateriale A, Lee KK, Huston CD. 2013. Drug repurposing screen reveals FDA-approved
inhibitors of human HMG-CoA reductase and isoprenoid synthesis that block Cryptosporidium
parvum growth. Antimicrob Agents Chemother 57:1804-14.
Soriano F, Santamaria M, Ponte C, Castilla C, Fernandez-Roblas R. 1988. In vivo significance of
the inoculum effect of antibiotics on Escherichia coli. Eur J Clin Microbiol Infect Dis 7:410-2.
Cohen BH, Saneto RP. 2012. Mitochondrial translational inhibitors in the pharmacopeia. Biochim
Biophys Acta 1819:1067-1074.
Ribes S, Pachon-Ibanez ME, Dominguez MA, Fernandez R, Tubau F, Ariza J, Gudiol F, Cabellos C.
2010. In vitro and in vivo activities of linezolid alone and combined with vancomycin and
imipenem against Staphylococcus aureus with reduced susceptibility to glycopeptides. Eur J Clin
Microbiol Infect Dis 29:1361-7.
Newton PN, Chaulet JF, Brockman A, Chierakul W, Dondorp A, Ruangveerayuth R, Looareesuwan
S, Mounier C, White NJ. 2005. Pharmacokinetics of oral doxycycline during combination
treatment of severe falciparum malaria. Antimicrob Agents Chemother 49:1622-5.
Garrabou G, Soriano A, Lopez S, Guallar JP, Giralt M, Villarroya F, Martinez JA, Casademont J,
Cardellach F, Mensa J, Miro O. 2007. Reversible inhibition of mitochondrial protein synthesis
during linezolid-related hyperlactatemia. Antimicrob Agents Chemother 51:962-967.
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M,
Soding J, Thompson JD, Higgins DG. 2011. Fast, scalable generation of high-quality protein
multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539.
Choi R, Kelley A, Leibly D, Hewitt SN, Napuli A, Van Voorhis W. 2011. Immobilized metal-affinity
chromatography protein-recovery screening is predictive of crystallographic structure success.
Acta Crystallogr Sect F Struct Biol Cryst Commun 67:998-1005.
Beebe K, Waas W, Druzina Z, Guo M, Schimmel P. 2007. A universal plate format for increased
throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities. Anal
Biochem 368:111-121.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.

8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
47.
48.
49.
50.
51.
Vinayak S, Pawlowic MC, Sateriale A, Brooks CF, Studstill CJ, Bar-Peled Y, Cipriano MJ, Striepen B.
2015. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature
523:477-80.
Arrowood MJ, Donaldson K. 1996. Improved purification methods for calf-derived
Cryptosporidium parvum oocysts using discontinuous sucrose and cesium chloride gradients. J
Eukaryot Microbiol 43:89S.
Love MS, Beasley FC, Jumani RS, Wright TM, Chatterjee AK, Huston CD, Schultz PG, McNamara
CW. 2017. A high-throughput phenotypic screen identifies clofazimine as a potential treatment
for cryptosporidiosis. PLoS Negl Trop Dis 11:e0005373.
Silva DG, J.R.; Ranade, Ranae; Herbst, Zackary; Nguyen, Uyen; Buckner, F.S.; Montanari, Carlos;
Gelb, Michael. 2017. A New Class of Antitrypanosomal Agents Based on Imidazopyridines. ACS
Med Chem Lett.
Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, Kotb M, Gillies SD, King M,
Mangada J, Greiner DL, Handgretinger R. 2005. Human lymphoid and myeloid cell development
in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.
J Immunol 174:6477-89.
Parr JB, Sevilleja JE, Samie A, Alcantara C, Stroup SE, Kohli A, Fayer R, Lima AA, Houpt ER,
Guerrant RL. 2007. Detection and quantification of Cryptosporidium in HCT-8 cells and human
fecal specimens using real-time polymerase chain reaction. Am J Trop Med Hyg 76:938-42.
Herbst ZM, Shibata S, Fan E, Gelb MH. 2018. An Inexpensive, In-House-Made, Microdialysis
Device for Measuring Drug-Protein Binding. ACS Med Chem Lett 9:279-282.
Scientific T. https://assets.thermofisher.com/TFS-
52.
53.
54.
55.
Assets/LSG/manuals/MAN0011619_SgleUse_RED_Plate_Insert_UG.pdf. Accessed
Spencer AC, Heck A, Takeuchi N, Watanabe K, Spremulli LL. 2004. Characterization of the human
mitochondrial methionyl-tRNA synthetase. Biochemistry 43:9743-9754.
8
87

8
88 Table 1. Protein sequence analysis of MetRS inhibitor-binding sites from different speciesa
Zone, pocket, or amino acid by sequence numberb
Pocket or species 247 248 249 250 287 289 290 291 292 456 460 461 470 471
472 473 474 476 477 478 480 481 519 522 523
Pocket
b
b
b
l
q
q
q
q
q
q
q
q
q
q
q
q
b
q
b
b
b
b
b
b
b
Trypanosoma
brucei
Pro Ile
Ala Ile
Pro Ile
Ala
Tyr
Tyr
Tyr
Tyr
Asp His
Asp His
Asp His
Asp Tyr
Gly
Gly
Gly
Gly
Gln Lys
Gln Lys
Leu Lys
Leu Ala
Ile
Val
Ile
Thr
Cys
Val
Thr
Val
Val
Tyr
Val
Val
Val
Val
Trp
Trp
Trp
Trp
Asp Ala
Asp Ala
Asp Ala
Asp Ala
Leu Asn Tyr
Leu Asn Tyr
Leu Asn Tyr
Ile
Ile
Ile
Phe His
Phe His
Phe His
Cryptosporidium
parvum/hominis
Ala
¶
Gly
Gly
Gly
Met Tyr
Homo sapiens
(mitochondrial)
Phe Tyr
Tyr
Ile
Tyr
Tyr
Homo sapiens
(
cytoplasmic)
Leu Pro
Thr
Ala
¶
Phe
Thr
Gly
Tyr
Asn Phe His
a
8
8
8
8
8
89
90
91
92
93
Sequence #s refer to the T. brucei sequence. UNIPROT accession codes: T. brucei - Q38C91; C. parvum – Q5CVN0; C. hominis – UPI0000452AB0; H. sapiens/mito -
Q96GW9; H. sapiens/cyto - P56192.
b
l = linker zone, b = benzyl pocket (methionine substrate pocket), q = quinolone pocket (auxiliary pocket formed upon inhibitor binding) .
¶ Ambiguous: due to different loop length could be Leu or His.
8
94
8
95
Table 2. Kinetic parameters of C. parvum MetRS compared to MetRS enzymes from other organisms
Met
ATP
Reference
MetRS
C. parvum
S.aureus
K
m
(µM)
71
K
cat (S-1
)
K
cat/K
m
(µM-1S-1
)
K
m
(µM))
K
cat (S-1
33
)
K
cat/K
m
(µM-1S-1
0.03
)
37
36
0.52
0.47
1040
347
500
85
This work
This work
(31)
77
26
0.07
S.aureus
100
18
25
0.25
25
0.05
Human mitoch
0.41
0.023
0.033
0.00038
(55)
8
96

8
97
Table 3. Activities of MetRS inhibitors with linear linkers (mean ± SD)
U. VT
C.
parvum
EC50
CRL-
8155
EC50
(µM)*
> 20.0
Hep
G2
EC50
(µM)*
> 20.0
COX-1
EC50
SDH-A
EC50
(µM)
C. parvum
MetRS Ki
(nM)
UW C.
parvum
(µM)
Molecule Name
Structure
EC50 (µM) (µM)
Reference
1
312
0.64 ± 0.01
18.55 ±
0.212 (n=2)
> 10.0;
50.0
2.43
>25
(24)
>
(n=2)
1
1
433
614
> 1.33
n =2)
> 20.00 ±
> 10.0,
11.2
14.7
12.2
14 ±
2.8
16.5 ±
4.9
(n=2)
(25)
(26)
(
0
.000 (n=2)
(n=2)
0.61 ± 0.06
n=2)
14.80 ±
2
7.60 ±
4.35 ±
0.92
(n=2)
31.5 ±
2.1 (n=2)
39.7 ± > 50.0
0.8
(
.404 (n=2) 0.74
n=2)
(n=2)
(
1
1
717
0.41 ± 0.06
n=2)
4.10 ± 2.59 5.17 ±
n=2) 0.45
1.04 ±
0.51
(n=2)
>25 (n=2) 14.1 ± 25.4 ±
(26)
(33)
(
9.5
n=5)
8.4
(n=4)
(
(
(n=2)
369 bumped
Not
applicable
2.4 ± 0.7
n=2)
Not
done
Not
Not
>40.0
>40.0
kinase inhibitor
Reference
standard)
applicable applicable
(
(

8
8
9
9
9
9
98
99
00
01
02
03
*Cytotoxicity data previously published for 1312 (24), 1614 (26),1717 (26), and 1369 (33)
Reference standard for CRL-8155 cells: Quinacrine EC50 = 3.99 ± 2.27 µM (n=14)
Reference standard for HepG2 cells: Quinacrine EC50 = 9.76 ± 2.73 µM (n=13)
Reference standard for COX-1: Chloramphenicol EC50 = 6.31 ± 0.91 (n=8)
Reference standard for SDH-A: Chloramphenicol EC50 = >50 ± 0 (n=8)
9
9
9
04
05
06

9
9
07
08
Table 4. Activities of MetRS inhibitors with a single ring in the linker (mean ± SD)
HO
MeO
O
N
N
Cl
N
N
H
Cl
MetRS inhibitor scaffold
Cmpd 2242
UW C.
Cl
SDH- CRL-
8155
Hep
G2
C. parvum
MetRS Ki
(nM)
parvum
EC50
(µM)
U. VT C.
parvum
EC50 (µM)
A
Molecule
Name
COX-1
EC50 (µM)
EC50 EC50 EC50
(µM) (µM)* (µM)*
>25 >50.0 >50.0
R1
R2
R3
R4
Reference
(27)
1
962
H
F
Cl
Cl
H
H
> 1.33 >20.000 ±
8.48 ± 0.81
(n=2)
>25
(n=2)
0
.000
n=2)
1.865 ±
.884
n=2)
0.060 ±
.011
n=2)
(
2
062
-CH
2
-CH
3
F
0.0162 ±
.0017 (n=2)
0.152 (n=2)
0.076 (n=2)
0.29
>25 >50.0 >50.0
(27)
0
0
(
2
2
114
067
-CH
-CH
2
-CH
-CH
3
3
F
-OCH
3
H
H
0.0023 +/-
.0011 (n=3)
0.075
0.042
>25 >50.0 >50.0
>25 >50.0 >50.0
(27)
0
0
(
2
Cl
Cl
Cl
0.0093 ±
.0002 (n=2)
0.408 ±
0.286
This work
0
(
n=2)
0.598
n=1)
2
2
091
093
-CH
-CH
2
-CH
-CH
3
3
Br
Cl
H
H
0.376 (n=2)
>50.0 >50.0
This work
(27)
(
2
-OCH
3
0.0009 +/-
0.036 ±
0.004
(n=2)
0.007 (n=2)
0.039
>25 >50.0 >50.0
0.0004
(n=12)
2
2
069
139
-CH
2
-CH
CH
2
-
3
F
F
Cl
Cl
H
H
0.016 +/-
0.010 (n=3)
0.411 ±
0.266
0.043 (n=2)
0.195
0.175
>25 >50.0 >50.0
This work
This work
(n=2)
-CH
2
2
-CF H
0.066 +/-
.022 (n=2)
0.284 ±
>25
33.3 >50.0
0
0
.128
(n=2)

2
2
2
259
138
258
-CH
-CH
-CH
2
2
2
-CF
-CF
-CF
2
2
2
H
H
H
F
Cl
Cl
-OCH
3
H
H
H
H
0.0038 +/-
.0014 (n=3)
0.134 ±
0.089
0.164
0.430
0.134
0.055
>25 >50.0 >50.0
>25 >50.0 >50.0
>25 >50.0 >50.0
This work
This work
This work
0
0
0
(n=2)
Cl
0.0187 +/-
.0004 (n=2)
0.454 ±
0.252
0.416 ±
0.127 (n=2)
(n=2)
-OCH
3
0.0036 +/-
.0019 (n=3)
0.19 ±
0.004
(n=2)
2
2
207
240
-CH
OH
-CH
OH
2
-
Cl
Cl
Cl
0.129
(n=1)
0.100 ±
0.019 (n=2)
>25 >50.0 >50.0
>50.0 >50.0
This work
This work
C(CH
C(CH
3
)
2
2
-
-OCH
3
-OCH
3
0.31 ±
0.170
3
)
2
(
n=2)
0.52 ±
.12
n=2)
2
2
080
242
-CH(CH
3
)
2
F
Cl
H
0.166
1.57
>25 >50.0 >50.0
>25 31139 >50.0
This work
This work
0
(
Structure
above
0.014 +/-
0.0048 (n=2)
0.67 +/-
0.19
(n=2)
9
9
9
9
9
9
9
9
09
10
11
12
13
14
15
16
*Cytotoxicity data previously published for 1962, 2062, 2093, and 2114 (27)
Reference standard for CRL-8155 cells: Quinacrine EC50 = 3.99 ± 2.27 µM (n=14)
Reference standard for HepG2 cells: Quinacrine EC50 = 9.76 ± 2.73 µM (n=13)
Reference standard for COX-1: Chloramphenicol EC50 = 6.31 ± 0.91 (n=8)
Reference standard for SDH-A: Chloramphenicol EC50 = >50 ± 0 (n=8)
9
9
9
17
18
19

9
20
Table 5. Physicochemical properties of ring-linker compounds (structures in Table 4)
Molecule
Name
*Protein
H-
bond
Solubility Solubility Solubility
binding PAMPA:
mouse Pe (pH
plasma: 7.4)
PAMPA:
Pe (pH
5.0)
Molecular
weight
limit @
limit at
pH 2.0
(uM)
limit @
pH 6.5
(uM)
Predicted
permeability
cm/s x 10^4
2.394
H-bond accept pH 7.4
donors ors
(g/mol)
log P
3.47
(uM)
% bound (nm/sec)
(nm/sec)
2
2
2
2
2
2
2
2
2
2
2
2
2
114
067
091
093
069
139
259
138
258
207
240
080
242
415.85
1
4
3
3
4
3
3
4
3
4
5
6
3
4
3
5
69.4
9.3
78.4
53.8
98.7
319
284
436.72
481.18
432.31
434.3
4.52
4.67
3.76
4.75
4.34
3.58
4.63
3.87
3.41
3.25
4.65
4.68
2.58
0.53
1
1
1
1
1
1
1
1
2
2
1
2
2
2
10.3
49
8.8, 9.8
<10
99.7
313
226
316
286
269
349
238
336
288
251
3.849
4.500
2.333
3.721
3.867
2.319
3.887
2.309
1.322
1.118
3.853
1.745
2.449
0.646
26.8 60.5, 67.2
39, 51.5
99.9
99.6
99.5
95.0
100.0
98.8
98.7
87.7
6
26.2
44.4
26.2
44.4
39.9, 49.1 36.4, 29.4
456.25
451.83
472.7
76.8
52
65
80.6
33
58.4
> 100
66.3
78.1
> 100
43.1
235
237
468.29
476.36
506.38
434.3
39.2
86.8
83.2
53.6
< 10
509.81
259.35
360.22
100.0
Propranolol
600
30
200
30
Methyclo-
thiazide
9
21
*Plasma protein binding for 2069 and 2093 were previously reported (27).

9
9
9
9
22
23
24
25
Table 6. Pharmacological properties of ring-linker compounds. In vitro metabolic
stability (T ) was measured in mouse and human liver microsomes. Pharmacokinetic
studies in mice (n=3 per compound) were done with a single PO dose at 50 mg/kg in
MMV vehicle (defined in Methods).
½
Mouse
Human
Mouse
Mouse Oral
PK: AUC ±
SD
Mouse Oral PK:
Concentrations
in pooled mouse
microsome microsome Oral PK:
stability
T1/2 (min)
Molecule
Name
stability
T1/2 (min)
Cmax ±
SD (µM)
(min*µmol/L) feces ± SD (µM)
2
114
7.7
15.2
7.5 ± 2.4
n=3)
1854 ± 103
(n=3)
11.4 ± 4.1 (n=3)
(
2
067
3.1
9.3
65.2 ± 17263 ± 5167
13.3 (n=3)
(n=3)
2
2
091
093
2
9.4
8.3
3.2
5.8 ± 1.54
1863 ± 658
(n=3)
3932 ± 431
(n=3)
31.1 ± 3.5 (n=3)
36.2 ± 3.7 (n=3)
(
n=3)
21.8 ± 8.8
n=3)
2
2
069
139
3.6
8.2
(
11.7
20.1
40.7 ± 10404 ± 2589
10.6 (n=3)
(n=3)
2
2
259
138
29.4
5.8
32.7
15
25.8 ± 16.2 (n=3)
43.6 ± 3.8 (n=3)
30.8 ± 1.3
6269 ± 1806
(n=3)
(n=3)
2
2
258
207
13.8
5.3
25.5
25.1
30.8 ± 6.5
n=3)
3048 ± 863
(n=3)
(
2
2
2
240
080
242
24.2
1.6
42.3
8
11.1 ±
1.8 (n=3)
856 ± 813
(n=3)
5092 ± 1466
(n=3)
846 ± 220 (n=3)
17.9 ± 5.4 (n=3)
52.7± 16.2 (n=3)
1
25.4 ± 8.6
n=3)
2.5 ± 0.7
n=3)
(
4.5
7.7
512 ± 51
(n=3)
(
9
26
*Microsome half-lives for 2093 and 2114 were previously published (27).