From Cells to Mice to Target: Characterization of NEU-1053 (SB-443342) and Its Analogues for Treatment of Human African Trypanosomiasis

Article abstract of DOI:10.1021/acsinfecdis.6b00202

Human African trypanosomiasis is a neglected tropical disease that is lethal if left untreated. Existing therapeutics have limited efficacy and severe associated toxicities. 2-(2-(((3-((1H-Benzo[d]imidazol-2-yl)amino)propyl)amino)methyl)-4,6-dichloro-1H-indol-1-yl)ethan-1-ol (NEU-1053) has recently been identified from a high-throughput screen of >42,000 compounds as a highly potent and fast-acting trypanocidal agent capable of curing a bloodstream infection of Trypanosoma brucei in mice. We have designed a library of analogues to probe the structure-activity relationship and improve the predicted central nervous system (CNS) exposure of NEU-1053. We report the activity of these inhibitors of T. brucei, the efficacy of NEU-1053 in a murine CNS model of infection, and identification of the target of NEU-1053 via X-ray crystallography.

Full text of DOI:10.1021/acsinfecdis.6b00202

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Article
From cells, to mice, to target: Characterization of NEU-1053 (SB-443342)
and its analogs for treatment of human African trypanosomiasis
William G. Devine, Rosario Díaz González, Gloria Ceballos-Perez, Domingo Isaac
Rojas-Barros, Takashi Satoh, Westley Tear, Ranae M. Ranade, Ximena Barros-
Álvarez, Wim G.J. Hol, Frederick S. Buckner, Miguel Navarro, and Michael P. Pollastri
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.6b00202 • Publication Date (Web): 22 Jan 2017
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From cells, to mice, to target: Characterization of NEUꢀ1053 (SBꢀ443342) and its
analogs for treatment of human African trypanosomiasis.
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William G. Devine,a Rosario DiazꢀGonzalez,b Gloria CeballosꢀPerez,b Domingo Rojas,b
Takashi Satoh,a Westley Tear,a Ranae M. Ranade,c Ximena BarrosꢀÁlvarez,d, e Wim G.
J. Hol,d Frederick S. Buckner,c Miguel Navarro,b and Michael P. Pollastria *
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aDepartment of Chemistry & Chemical Biology, Northeastern University 360 Huntington Avenue,
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Boston, MA USA; Instituto de Parasitología y Biomedicina "López-Neyra", Granada 18100
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Spain; Departments of Medicine, and Biochemistry, University of Washington, Seattle, WA
98195 USA; eLaboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de
los Andes, Mérida, Venezuela.
ABSTRACT
Human African Trypanosomiasis is a neglected tropical disease that is lethal if left
untreated. Existing therapeutics have limited efficacy and severe associated toxicities.
2ꢀ(2ꢀ(((3ꢀ((1HꢀBenzo[d]imidazolꢀ2ꢀyl)amino)propyl)amino)methyl)ꢀ4,6ꢀdichloroꢀ1Hꢀindolꢀ
1ꢀyl)ethanꢀ1ꢀol (NEUꢀ1053) has recently been identified from a high throughput screen
of >42,000 compounds as a highly potent and fast acting trypanocidal agent capable of
curing a blood stream infection of T. brucei in mice. We have designed a library of
analogs to probe the SAR and improve the predicted CNS exposure of NEUꢀ1053. We
report the activity of these inhibitors of Trypanosoma brucei, the efficacy of NEUꢀ1053 in
a murine CNS model of infection, and identification of the target of NEUꢀ1053 via Xꢀray
crystallography.
KEYWORDS
Trypanosoma brucei, methionylꢀtRNA synthetase, medicinal chemistry
INTRODUCTION
Insectꢀborne trypanosomal diseases are a menace to human health. Human African
trypanosomiasis (HAT, or sleeping sickness) is a neglected tropical disease caused by
two subspecies of Trypanosoma brucei for which current therapeutics are toxic and
inconvenient. Though there are two compounds in clinical trials, SCYXꢀ71581 and
fexinidazole,2 given the clinical failure rate for infectious diseases,3 it is prudent to
continue the search for new drugs.
To that end, we recently described a highꢀthroughput screening campaign performed as
part of an industryꢀacademic partnership between GlaxoSmithKline, Spanish National
Research Council (CSIC), and Northeastern University, in which we uncovered 798
inhibitors of T. brucei cellular proliferation. We also reported in vitro drug metabolism,
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physicochemical properties, and pharmacokinetics data, plus kinase selectivity data for
key analogs.4 Included in that report was NEUꢀ1053 (SBꢀ443342, 1), a singleton
compound identified in the screen that showed rapid and irreversible proliferation
inhibition of T. brucei, showed good plasma exposure, and cured a bloodstream
infection in a T. brucei rhodesiense mouse model of HAT. As a singleton hit with no
other analogs included in the screening campaign, there was no SAR information
apparent from the HTS. Besides looking to better understand the SAR of this series, we
wished to explore the various structural regions of the compound in order to identify
effective analogs with more attractive physicochemical properties. We report those
efforts here, definitively demonstrate the mechanism of action for 1, and describe the
results of an in vivo efficacy experiment for this compound in a murine model of Stage II
HAT.
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RESULTS
The overall strategy for the SAR exploration of 1 is shown in Figure 1. First, noting that
the chlorine atoms on the indole provide substantial contribution to molecular weight
and lipophilicity, we were interested to understand their importance. Second, the
hydroxyethyl substituent on the indole nitrogen seemed to be a potential metabolic
liability, and exploration of this region was needed. Third, the linker between the two
aromatic systems in the molecule needed to be probed in terms of length, vector, and
rigidity. Lastly, looking to reduce the size of the molecule, we wished to better
understand the requirements of the 2ꢀaminobenzimidazole eastern end by replacing the
benzimidazole functionality. We first describe the synthesis of these analogs, and will
then discuss the impact on potency and properties for this series.
Figure 1. 1 SAR regions of interest
The synthesis of 1 and its 3,5ꢀdichloroindole analogs commenced with the condensation
of hydrazine 2 and ethyl pyruvate to yield a mixture of E and Z isomers of 3 (Scheme
1). Cyclization of 3 generated 3,5ꢀdichloroindole 4, which could be recrystallized from
H2O/ethanol. Alkylation with the appropriate alkyl halides or tosylates5, 6 and subsequent
reduction with DIBAL gave corresponding Nꢀsubstituted 2ꢀ(hydroxymethyl)indoles 6aꢀf
in good to high yields. Oxidation of the benzylic alcohol with freshly prepared MnO2
following the Attenburrow procedure7 produced aldehydes 7aꢀf in 50ꢀ71% yield. The
oxidation of diol 6a also generated lactone 8 in 20% yield, likely stemming from overꢀ
oxidation of the intermediate lactol. This material could be reverted to 6a via reduction
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or used for further analog synthesis (vide infra). The nonꢀhalogenated 13 was
synthesized in a similar manner from the commercially available indole 9 (Scheme 2).
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Scheme 1. Synthesis of the 3,5ꢀdichloroindole western end of 1 and its analogs.
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Reagents and conditions: (a) ethyl pyruvate, EtOH, reflux, 1 hr; (b) PPA, 130 °C, 10 min; (c) methyl
bromoacetate, K2CO3, DMF, rt, o.n.; (d) NaH, DMF, 0 °C, 30 min; then CH3I, 0 °C→rt, 3 hrs; (e) NaH,
DMF, 0 °C, 30 min; then CH3OCH3Cl , 0 °C→rt, 3 hrs; (f) NaH, DMF, 0 °C, 30 min; then CH3OCH2CH2Br ,
0 °C→rt, 18 hrs; (g) K2CO3, DMF, BocNH(CH2)2OTs, rt, o.n.; (h) DIBAL, THF, rt, 1 hr; (i) MnO2, THF, rt, 2
hrs.
Scheme 2. Synthesis of the western end of dechloroꢀ1 analogs.
Reagents and conditions: (a) H2SO4, EtOH, reflux, o.n.; (b) methyl bromoacetate, K2CO3, DMF, rt, 3 hrs;
(c) LiAlH4, THF, 0 °C, 30 min, rt, 1.5 hrs; (d) MnO2, CH2Cl2, rt, o.n.
The alcohol moiety of compound 6b was protected as the TBS ether, and the
sulfonylation of this compound was performed using the same protocol for 5cꢀf; this
reaction also effected desilylation, providing 16 in 31% yield (Scheme 3). The free
alcohol was oxidized to the corresponding aldehyde with MnO2 in modest yield.
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Scheme 3. TBS protected Nꢀsubstituted indole synthesis.
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Reagents and conditions: (a) TBSCl, imidazole, CH2Cl2, rt, 1 hr; (b) NaH, DMF, rt, 30 min; CH3SO2Cl, rt, 1
hr; (c) MnO2, CH2Cl2, rt, 2 hrs.
Aminoalkylꢀ2ꢀaminobenzimidazole intermediates were prepared as shown in
Schemes 4ꢀ7. Briefly, 20 was formed via heating of 18 in the presence of urea followed
by deoxyꢀchlorination with POCl3. MonoꢀBoc protected diamines 22aꢀd, 26aꢀb, 29, and
32, prepared according to known procedures8ꢀ13 or commercially available, were
coupled to 20 using microwave irradiation at 150 °C. Removal of the Boc group was
effected under either acidic conditions to yield the bisꢀTFA salt, or via microwave
promoted thermolysis to give the free base. Attempts to couple 20 with 21a directly
were successful in generating product, though the purification of 24a was complicated
due to its high water solubility. This was overcome by the introduction a Boc group,
allowing purification via extraction followed by chromatography. The isolation of 24 was
accomplished by simple concentration of the reaction medium.
Scheme 4. Chloroꢀ1Hꢀbenzo[d]imidazole synthesis
Reagents and conditions: (a) urea, (CH2OH)2, 140 °C, 1 hr, 170 °C, 7 hrs; (b) POCl3, reflux, 2 hrs.
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Scheme 5. Acyclic eastern half synthesis
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Reagents and conditions: (a) Boc2O, CHCl3, 0 °C→rt, o.n.; (b) 20, toluene, ꢁW, 150 °C, 6 hrs; (c) NaH,
DMF, CH3I, rt, 1 hr; (d) TFA, CH2Cl2, rt, o.n.; (e) H2O, ꢁW, 150 °C, 1 hr
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Scheme 6. 1 cyclohexyl linker replacement synthesis
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Reagents and conditions: (a) Boc2O, CH2Cl2, 0 °C→rt, o.n.; (b) 20, toluene, ꢁW, 150 °C, 6 hrs; (c) TFA,
CH2Cl2, rt, o.n.
Scheme 7. 1 cyclic linker analog synthesis
Reagents and conditions: (b) 20, toluene, ꢁW, 150 °C, 6 hrs; (b) TFA, CH2Cl2, rt, o.n.
2ꢀ(aminophenyl)aminobenzimidazoles 36aꢀc were prepared and purified without
Boc protection under microwave heating (Scheme 8). Reaction times were reduced to
15 minutes by increasing the temperature from 150 °C to 200 °C.
Scheme 8. Phenylenediamine linker synthesis
Reagents and conditions: (a) 20, xylenes, ꢁW, 200 °C, 15 min.
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Reductive amination of 2ꢀindolecarboxaldehydes 7aꢀf, 13, and 17 with 24a was
carried out with NaBH3CN under either mildly basic conditions with the bisꢀTFA salt of
24a, or mildly acidic conditions with the free base (Scheme 9, product structures in
Tables 1 and 3). The same reaction conditions were used to couple 7a with amines 24,
28, 31, 34, and 36 (Table 4).
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Scheme 9. Synthesis of 1 analogs via reductive amination
Reagents and conditions: (a) amine, KOAc, NaBH3CN, CH3OH; (b) amine, AcOH, NaBH3CN, CH3OH
The synthesis of analogs 38, substituting the dichloroindole scaffold with
chlorophenyl rings, are shown in Scheme 10, via reductive amination from
commercially available chlorobenzaldehydes 37.
Scheme 10. Synthesis of chlorophenyl 1 analogs
Reagents and conditions: (a) 24a, AcOH, NaBH3CN, CH3OH
The side product 8 (Scheme 1) was used to produce 39 via ester aminolysis with
the free base of 24a and microwave irradiation (Scheme 11).
Scheme 11. Synthesis of 38 via esterꢀamide exchange
Reagents and conditions: (a) 24a, DMF, ꢁW, 150 °C, 1 hr
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To synthesize analogs varying the benzimidazole of 1, aldehyde 7a was first reductively
aminated with 22a (Scheme 12). Exchange of the Boc protecting group for the acidꢀ
stable Nꢀ(1ꢀ(4,4ꢀdimethylꢀ2,6ꢀdioxocyclohexylidene)ethyl) (Dde) protecting group
provided 41 in high yield. The secondary amine was Boc protected and the enamine
removed upon heating in the presence of hydrazine monohydrate. The free primary
amine of 43 was then reacted with phenylisocyanate to provide 44a, 2ꢀ
chlorobenzoxazole (44b), or 4ꢀchloropyrimidine (44c). Deprotection of the Bocꢀ
protected secondary amines under acidic conditions gave the final analogs in modest
yields.
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Scheme 12. Heterocyclic replacements on the eastern half of 1
Reagents and conditions: (a) 22a, AcOH, NaBH3CN, CH3OH, rt, 12 hr; (b) 3M aq. HCl, THF, rt, 1 hr, 80
°C, 1 hr; then DDEꢀOH, (iꢀPr)2NEt, CH2Cl2, CH3OH, rt, 12 hr; (c) Boc2O, K2CO3, CH3CN, H2O, rt, 12 hr; (d)
H2NNH2—H2O, CH3CN, 45 °C, 3 hr; (e) PhCNO, CH3CN, rt, 12 hr; (f) 2ꢀchlorobenzoxazole, (iꢀPr)2NEt,
CH3CN, 80 °C, 12 hr; (g) 4ꢀchloropyrimidine, (iꢀPr)2NEt, CH3CN, ꢂW, 150 °C, 2 hr; (h) 4M HCl in 1,4ꢀ
dioxane, rt, 6 hr.
In vitro assessment against T. brucei.
Indole halogens. Removal of both chlorides on the indole ring resulted in an 78x
loss in potency (46, EC50: 75 nM, Table 1). A significant reduction in MW (432→364)
and clogP (3.60→2.39) was effected however, resulting in the highest CNS
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multiparameter optimization (MPO) score14 among these analogs. CNS exposure is
essential to effectively treat stage II HAT when the parasites have invaded the CNS and
crossed the blood brain barrier (BBB). Noting that a CNS MPO score ≥4 is suggestive of
CNS penetration, 46 score was calculated to be 4.3. This compound also retained a
high lipophilic ligand efficiency (LLE, pEC50ꢀcLogP) of 4.71 (the desirable range for LLE
is ≥4).15 Replacement of the aminomethyl group of 1 with an amide (39) resulted in a
520ꢀfold loss in activity. This correlates well with the conclusions from the linker SAR in
that the basicity of the dialkylamine is essential for high potency.
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Table 2 shows the results of replacing the dichloroindole scaffold with simplified
chlorophenyl rings. Compound 38c, retaining the 3,5ꢀdichloro motif showed only a slight
reduction in potency (EC50=16 nM). Removal of one of the chlorine atoms (38a) resulted
in an 8ꢀfold loss in activity (EC50=0.15 ꢂM) compared to 38c and migration of the
chloride to the para position (38b) resulted in a further 6ꢀfold loss in activity (EC50=0.96
ꢂM). Our observation that the indole moiety in this class of compounds can be replaced
with a simple dichlorophenyl group may provide a useful path forward in the event that
toxicity due to indole metabolism is observed.
Indole N-substituents. Modifications to the 2ꢀhydroxyethyl group of 1 displayed mixed
results (Table 3). Truncations (47a, 47b) or a shift of the oxygen atom to an internal
position (47c) resulted in less than a 10ꢀfold loss in activity. Larger groups (47d, 47e,
47f) comparatively caused a far larger loss in activity (>300x). Replacement of the
alcohol with an amine (47g) led to around a 200ꢀfold loss in activity. MPO scoring for
these compounds was similar (≤∆ 0.3) to 1 except for the large increase resulting from
addition of the methanesulfonamide group (47e, MPO=4.1) and large decrease
apparent in the Bocꢀprotected 47f (MPO=1.5). Generally, the smaller indole Nꢀ
substituents remained highly active while those that increased steric bulk resulted in a
drop in potency by 2ꢀ3 orders of magnitude.
Linker. Contracting or extending the diamino linker by a methylene unit (48a and
48b) did not lead to a loss in potency (EC50=5.0 nM each, Table 4) compared to 1.
Compound 48a did however possess a slightly improved MPO score (3.8). Capping
both amines of the linker with methyl groups was not tolerated (48c, EC50=0.98 ꢂM).
The trimethylated compound (48d) showed approximately an additional 3ꢀfold loss in
activity (EC50=2.2 ꢂM).
The tolerance for a chain of 2ꢀ4 carbons between amines suggests adequate
space within the biological target(s) of action for conformational variations of the linker.
The paraꢀ, metaꢀ, and orthoꢀ phenylenediamine linkers, designed to restrict the
conformation of the linker, all showed a dramatic loss in activity. The ortho-linked 48e
was the most active (EC50=0.42 ꢂM), followed closely by the meta-linked 48f
(EC50=0.96 ꢂM). The paraꢀlinked compound, 48g, was an additional order of magnitude
less active (EC50=5.5 ꢂM). These data suggested either that (1) a bent orientation of the
linker is closest to the active conformation of 1, or (2) the basicity of these nitrogens is
important.
Compounds 48hꢀk further probed the tolerance for reduced linker flexibility and
diamine orientation. Three of the four saturated cyclic linked analogs, 48hꢀj, remained
subꢀmicromolar in activity. Compounds 48h and 48i were within 2ꢀfold of one another,
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showing no preference for cis or trans substitution of the cyclohexyl ring, and were
essentially equipotent to the aromatic analog 48e. Compound 48k, bearing a piperazinyl
linker, was >5 ꢂM, compared to the 4ꢀaminopiperadinyl linked 48j (EC50=0.40 ꢂM). This
difference in activity is likely not a result of linker reduction as 48a retained essentially
all activity compared to 1. Rather, taken together with the poor activity of 48c and 48d,
the reduced activity of 48k illustrates the importance of (1) the hydrogen bonding donor
motif and (2) a high degree of flexibility in the linker for high potency.
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Benzimidazole replacements. Any replacements for the benzimidazole ring that
lacked a hydrogen bond donor were not tolerated (Table 5, 45b and 45c). Specifically,
changing to an isosteric benzoxazole (45b) led to around an 800ꢀfold loss in activity
compared to 1. Compound 45c, also devoid of a hydrogen bond donating 4ꢀpyrimidine
ring, possessed activity approximating 45b (0.79 ꢂM vs. 0.27 ꢂM). Reintroduction of a
hydrogen bond donor with a urea NꢀH (45a), which could recapitulate that of the
benzimidazole, regained the high potency observed for 1.
All analogs of 1 were screened against MRC5ꢀSV2 human lung cells to assess
toxicity. Most compounds possessed a selectivity index in the 10ꢀ50x range.
Compounds in the single digit nanomolar range against T. brucei displayed a higher
selectivity index in the range of 900ꢀ7,000x.
Physicochemical and ADME properties. The calculated physicochemical
properties of compounds 1, 38aꢀc, 39, 45aꢀc, 46, 47aꢀg, and 48aꢀk are presented in
Table 6, color coded in terms of desirability. Compound 47e is noteworthy among
analogs replacing the 2ꢀhydroxyethyl group for its high MPO score above 4.0, due to its
reduced clogP and pKa (calculated using JChem for Excel, Chemaxon, Inc.) Analogs
48g, 48f, 48e, 48h, and 48i bearing phenylenediamine and 1,2ꢀdiaminocyclohexyl
linkers possess poor MPO scores as a result of high molecular weights, clogP, and
clogD values.
An excellent MPO score (4.3) is calculated for 46, which resulted from reduced
molecular weight compared to 1 (363.5, ∆68.9), clogP (2.39, ∆1.21), and clogD (0.36,
∆1.23); this results simply from the removal of the chlorine atoms from the indole. In
addition to the improved MPO scores of many compounds in this series, several
compounds retained LE values ≥0.30 and LLE values ≥4.0, indicating a good balance of
size and lipophilicity.
In vitro absorption, distribution, metabolism, and excretion (ADME) properties
were collected for several compounds and are tabulated in Table 7. Few compounds
improved the aqueous solubility to an appreciable extent, with 46 as the only significant
exception at 298 ꢁM. Compound 46 was also the only analog to show reduced human
plasma protein binding (PPB) to <99%, or to reduce the logD7.4 below 3.0 among those
tested. Of the 16 compounds assessed for human liver microsome clearance (HLM
Clint), only 4 analogs possessed lower clearance than 1. Compounds 47a and 47b
displayed the lowest human microsomal clearance, implicating the 2ꢀhydroxyethyl group
may indeed be a metabolic handle as we expected. Others with high microsomal
clearance are 47d, 47e, 47f, 48c, 48e, and 48h; the Boc protected analog 47f and the
dimethylamino analog 48c both showing rapid metabolism (142 and 135 ꢁL/min/mg
respectively). Similarly, few compounds possessed good rat hepatocyte clearance, with
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only 47g (Clint = 7.96 ꢁL/min/106) and 48k (Clint = 12.8 ꢁL/min/106) showing any
significant improvement over 1.
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Pharmacokinetics study. Though we previously reported plasma pharmacokinetics, we
did not determine CNS drug levels. Thus, female BALB/c mice were injected with a
single, 10 mg/kg intraperitoneal dose of 1, and plasma and brain levels were measured,
showing a brainꢀtoꢀplasma ratio of 0.39, with drug levels of 31.6 ng/g of brain tissue
measured at the 24 hour time point. (The PK parameters are summarized in the
Supporting Information, Tables S1ꢀS3 and Figure S1). With this evidence in hand, we
proceeded to assess this compound in a mouse model of Stage II HAT.
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Murine CNS infection model. We advanced 1 to a mouse model of a CNS infection with
T. b. brucei (GVR 35 strain). On day 0, 2 groups of 5 mice each were infected with T. b.
brucei and checked for parasitemia on day 14. One group, taken as the control, was
treated with a single dose of 40 mg/kg Berenil on day 21. From days 22 to 25 the
control group was treated with the vehicle DMSO in PBS. The other group of mice was
treated with 1 at a dose of 20 mg/kg/day on days 21ꢀ25. Following a 2ꢀday hiatus,
treatment with 1 was resumed on days 28ꢀ32. Parasitemia was checked from both
groups via examination of tail blood on days 14, 28, 35, 39, and 43. The results are
reported in Figure 2 and Table Sꢀ4.
Figure 2. Mice without parasitemia in a mouse model of CNS infection with T. b. brucei
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Berenil control group
Group treated with 1
Limit of detection for parasitemia = 125,000 parasite/mL blood.
On day 28, 2 days after the 1st regimen of treatment was completed, 3/5 mice
treated with 1 showed no detectable parasitemia. Following the 2nd round of treatment,
this number increased to 4/5 mice showing no detectable parasitemia on day 35. When
checked on day 39 however, all mice treated with 1 showed a full relapse of parasites in
the blood. The control group comparatively showed no detectable levels of parasitemia
when checked on days 28, 35, and 39 in all 5 mice, and only 2 mice showing a relapse
on day 43. Since recrudescence was observed even in animals where the bloodstream
infection was cleared by 1, we interpret this result to suggest that compound 1 does not
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have sufficient antiparasitic activity in the brain to cure Stage 2 HAT. This may be
attributed in part to the high PPB (>99.9%) and rapid clearance (rat hepatocyte
Clint=24.9 μL/min/106 cells) of 1.
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Target Identification. Though the potent activity of 1 was uncovered from a set of
compounds selected on the basis of their likely kinase inhibition activity, we noted that
the structure of 1 was quite similar to a class of previously reported T. brucei methionylꢀ
tRNA synthetase (MetRS) inhibitors (such as compound 1312, Figure 3).16 T. brucei
has a single MetRS and is necessary for cell growth as previously reported using RNA
interference.16 We wished to determine whether 1 inhibited T brucei MetRS.
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Figure 3. Structures of previously published T. brucei MetRS inhibitors (131216, 143317, and MetꢀSA118)
Biochemical assessment. A functional assay for the T. brucei MetRS enzyme based on
ATPꢀdepletion was previously described.19 Compound 1 was determined to be a highly
potent inhibitor, with an IC50 below the lower detection limit of the assay (<25 nM)
(Figure 4). The positive control compound, MetꢀSA118 Figure 3, is also a potent inhibitor
with an IC50 of <25 nM.
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Figure 4. T. brucei MetRS inhibition assay. IC50s of both the positive control, MetꢀSA1, and compound 1
are <25 nM (the lower detection limit of the assay as described in the results and discussion). Error bars
represent standard error of the mean of three replicates. Pentamidine, a negative control, had no
T. brucei MetRS inhibition up to 10 ꢁM (results not shown).
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MetꢀSA1
compound 1
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Figure 5. Wildꢀtype and 1433ꢀresistant T. brucei growth inhibition assays. Results from two independent
assays, as indicated with the 1 and 2. Error bars represent standard error of the mean of three replicates.
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WT 1
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Potency shift in resistant parasites. To further test if compound 1 is targeting the T.
brucei MetRS, a T. brucei growth inhibition cell assay was conducted with a strain that
is resistant to MetRS inhibitors. The resistant strain was generated by serial in vitro
passage of wildꢀtype T. brucei with the MetRS inhibitor 1433 (Figure 3) and was shown
to have ~35ꢀfold upregulated expression of MetRS mRNA.18 The EC50 of 1 and 1433 are
~107ꢀfold and ~48ꢀfold less potent against the 1433ꢀresistant strain compared to the
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wildꢀtype (Figure 5 and Table 8), consistent with the hypothesis that 1 acts by inhibiting
the MetRS enzyme. Pentamidine does not inhibit T. brucei MetRS (data not shown) and
does not show a difference in EC50 values between the 1433ꢀresistant and wildꢀtype
strains.
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Structural biology. Even though crystals of TbruMetRS•Met would disintegrate after ~20
seconds in contact with a solution of 1, it was possible to replace the bound methionine
with 1 in the binding site using short soaking times.20 The structure in complex with 1
was determined at 2.6 Å resolution and deposited in the Protein Data Bank with PDB
identifier 5TQU. Data collection and refinement statistics are shown in Table S5
(supporting information) and the electron density map for the compound is shown in
Figure 6A. Compound 1 binds to TbruMetRS in a manner similar to previouslyꢀreported
compoundꢀbound structures.20, 21 Characteristically, two pockets are involved in binding
of the inhibitor (Figure 6B): the enlarged methionine pocket (EMP), formed mainly by
hydrophobic residues including the ones engaged in methionine binding, and the
“auxiliary pocket” (AP), not present in the methionine bound state.20 The EMP, formed
mainly as a result of the movement of TbruMetRS residues Val473, Trp474 and
Phe522, is occupied by the 4,6ꢀdichloroindolylꢀring moiety which also interacts with
residues Ile248 and Tyr481. The AP is occupied by the benzimidazole moiety which
interacts with His289, Gly290 and Val473, while establishing a hydrogen bond with
Asp287 through one of the nitrogens of the benzimidazole ring with a distance of 3.0 Å.
The flexible linker of 1 forms hydrophobic interactions with the aromatic group of residue
Tyr250 and a hydrogen bond through the exocyclic amine of the benzimidazole moiety
with the catalytic Asp287 with a distance of 2.8 Å (Figure 6C). In a novel interaction
pattern for TbruMetRS inhibitors, the OH group in the hydroxyethyl substituent of the
4,6ꢀdichloroindolylꢀring moiety makes a hydrogen bond with residue Asp287 with a
distance of 2.6 Å. As a result, Asp287 plays a central role in establishing polar contacts
with the three different regions of 1: the linker, the benzimidazole moiety and the OH
group in the hydroxyethyl moiety. Furthermore, at least one water molecule is part of
what seems to be a crucial hydrogen bonding network involving Asp287 and 1 (Figure
6C).
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When comparing the new structure to previously reported TbruMetRS complexes
with inhibitors bound, the overall binding mode of 1 is quite similar. Taking the
TbruMetRS•1331 structure (Figure 6D) as a representative of previously determined
inhibitor bound structures, it appears that the two ring systems of each inhibitor occupy
similar positions. For instance, the two chlorine atoms are only 0.8 and 0.3 Å (Clꢀ4 and
Clꢀ6, respectively) apart in the two structures (Figure 6E) and the linkers follow a similar
path. The key difference is the extra interaction of the hydroxyl group of 1 with Asp287
(compare Figures 6C and 6f), representing one additional hydrogen bond between the
inhibitor and the target enzyme.
CONCLUSIONS
In summary, we report the structureꢀactivity relationships for the various regions of 1,
and described its activity in a mouse model of CNS infection of T. brucei. In addition, the
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compound’s mechanism of action via T. brucei MetRS was conclusively elucidated by a
combination of biochemical, cellular, and crystallographic techniques. Despite the
excellent in vitro antiꢀparasitic potency of 1, the lack of CNS activity in the lateꢀstage
model of HAT necessitates further optimization of CNS permeability and
pharmacokinetics; this work is ongoing.
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Figure 6. Binding of 1 to TbruMetRS. (A) The TbruMetRS•1 structure (PDB: 5TQU) with the difference
electron density map calculated by omitting the inhibitor, contoured at the 3σ level (positive density in
grey, negative density in red). (B) General features of the 1 binding mode. The position of the methionine
sulfur atom in the TbruMetRS•methionine structure (PDB: 4EG1)20 is shown with a yellow cross. The
protein surface and the two pockets, EMP and AP, where the inhibitor is bound are shown. (C) Hydrogen
bond network in the TbruMetRS•1 structure. The label for residue Asp287 is highlighted with a rectangle.
The hydroxyethyl substituent of the 4,6ꢀdichloroindolylꢀring moiety establishes an extra hydrogen bond
with residue Asp287. (D) Chemical structures of compound 1 and the previously reported TbruMetRS
inhibitor 1331. (E) Superposition of compounds 1 and 1331 (PDB: 4EG7)20 bound to TbruMetRS. (F)
Hydrogen bond interactions between inhibitor and enzyme in the TbruMetRS•1331 structure.
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A
C
B
6-Cl
EMP
4-Cl
AP
linker
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S of
F522
W47
D
V473
1
2.8 Å
Y48
3.0 Å
1331
2.6 Å
water
2.6 Å
G29
H289
D287
I248
F522
W47
E
F
V473
2.6 Å
Y48
3.0 Å
G29
H289
D287
I24
ACKNOWLEDGMENTS
Research reported in this publication was supported by the National Institute of Allergy
and Infectious Diseases of the National Institutes of Health under award numbers
R56AI099476 (to MPP), R01AI114685 (to MPP and MN), R01AI084004 (to WGJH) and
R01AI097177 (to FSB and RMR). We acknowledge the support of a Fulbright
Fellowship to XBꢀA. Funding from Northeastern University is also acknowledged. We
wish to thank Erkang Fan (University of Washington) for providing compounds 1433 and
MetꢀSA1. We thank Christophe L. M. Verlinde for his contributions to the structure
validation process. We thank Stewart Turley and Robert Steinfeldt for providing support
for the Xꢀray data collection and computing environment at the Biomolecular Structure
Center of the University of Washington. Crystallography performed in support of the
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work benefitted from remote access to resources at the Stanford Synchrotron Radiation
Lightsource supported by the U.S. Department of Energy Office of Basic Energy
Sciences under Contract No. DEꢀAC02ꢀ76SF00515 and by the National Institutes of
Health (P41GM103393).
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SUPPORTING INFORMATION
Experimental details for the pharmacokinetics experiments, chemical syntheses, Xꢀray
crystallography, and in vitro and in vivo biological methods are available in the
Supporting Information. In addition, annotation of compounds with NEU registry
numbers and SMILES strings are provided. This information is available free of charge
via the Internet at http://pubs.acs.org/.
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R.; Koh, C. Y.; Brown, S.; Lohse, J.; Verlinde, C. L.; Fan, E.; Bannister, T.; Scampavia, L.; Hol,
W. G.; Spicer, T.; Hodder, P. Identification of potent inhibitors of the Trypanosoma brucei
methionylꢀtRNA synthetase via highꢀthroughput orthogonal screening. J Biomol Screen 2015,
20, 122ꢀ130.
20.
Koh, Cho Y.; Kim, Jessica E.; Shibata, S.; Ranade, Ranae M.; Yu, M.; Liu, J.; Gillespie,
J. R.; Buckner, Frederick S.; Verlinde, Christophe L. M. J.; Fan, E.; Hol, Wim G. J. Distinct
States of MethionylꢀtRNA Synthetase Indicate Inhibitor Binding by Conformational Selection.
Structure 2012, 20, 1681ꢀ1691.
21.
Koh, C. Y.; Kim, J. E.; Wetzel, A. B.; de van der Schueren, W. J.; Shibata, S.; Ranade,
R. M.; Liu, J.; Zhang, Z.; Gillespie, J. R.; Buckner, F. S.; Verlinde, C. L. M. J.; Fan, E.; Hol, W.
G. J. Structures of Trypanosoma brucei MethionylꢀtRNA Synthetase with UreaꢀBased Inhibitors
Provide Guidance for Drug Design against Sleeping Sickness. PLoS Neglected Tropical
Diseases 2014, 8, e2775.
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TABLES
8
9
Table 1. Activity of modified indole analogs of 1 against T. brucei
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MRC5ꢀSV2
T. brucei
Selectivity
(TC50/EC50)
Compound
R
X
EC50 (nM)a
TC50 (μM)b
1
Cl
Cl
H
CH2
CO
0.96
12
18
39
46
500
36
CH2
75
27c
360
a SD <0.20. b SD <1.5 unless otherwise noted. cSD=9.3
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Table 2. Activity of chlorophenyl analogs of 1 against T. brucei
5
6
7
8
9
MRC5ꢀSV2
T. brucei
Selectivity
(TC50/EC50)
Compound
38a
R
EC50 (nM)a
TC50 (μM)b
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
150
960
22
147
23
38b
22
22
38c
16
1375
a SD <0.20. b SD <3.0.
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Table 3. Activity of indole Nꢀsubstituted analogs of 1 against T. brucei
5
6
7
8
9
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
T. brucei
MRC5ꢀSV2
Selectivity
(TC50/EC50)
R
H
Compound
47a
EC50 (nM)a
TC50 (ꢁM)b
9.0
5.0
8.8
978
1960
925
16
47b
CH3
9.8
7.4
10
47c
8.0
47d
620
340
790
47e
1.4
8.1
8.4
4.1
10
47f
47g
200
42
a SD <0.20. b SD <3.0.
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Table 4. Activity of linker analogs of 1 against T. brucei
5
6
7
8
9
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MRC5ꢀSV2
T. brucei
Selectivity
(TC50/EC50)
Compound
48a
R1
R2
EC50 (nM)a
TC50 (μM)b
H
5.0
5.0
26c
11
22
10
25
5200
2200
22
48b
48c
48d
48e
H
H
980
2200
420
CH3
H
4.5
60
48f
H
960
21
22
48g
48h
H
H
5500
680
23
14
4.2
21
48i
48j
H
H
H
340
400
17
12
50
50
30
ꢀꢀ
48k
>5000
a SD <0.55. b SD <3.0 unless otherwise noted. c SD=8.8
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Table 5. Activity of chlorophenyl analogs of 1 against T. brucei
6
7
8
9
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MRC5ꢀSV2
T. brucei
Selectivity
(TC50/EC50)
Compound
45a
R
EC50 (nM)a
TC50 (μM)b
2.0
14
7000
45b
45c
790
26
33
ꢀꢀ
270
>50
a SD <0.20. b SD <3.0.
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Table 6. Physicochemical properties of 1 and its analogs
5
6
7
8
MW
Hꢀbond
donorsa
TPSA
MPO
Cmpd
clogPa,c clogDa,d
pKab,c
LE
LLE
(g/mol)a
(Ų)b,c Scoree
1
432.4
314.8
314.8
349.3
446.3
435.4
433.3
394.3
363.5
388.3
402.3
432.4
446.4
466.4
531.5
431.4
418.3
446.4
460.4
474.4
466.4
466.4
466.4
472.4
472.4
458.4
444.4
3.60
3.44
3.44
4.04
3.11
3.28
3.66
2.30
2.39
4.06
4.29
4.35
4.24
2.88
4.98
3.49
3.54
4.11
4.62
4.84
5.34
5.34
5.34
4.95
4.95
3.93
4.38
1.59
1.57
1.53
2.33
2.98
1.36
1.78
0.42
0.36
2.06
2.28
2.36
2.23
2.08
2.97
ꢀ0.73
1.88
1.82
3.43
3.66
5.07
5.06
5.04
2.94
2.94
3.61
4.30
4
3
3
3
4
4
3
3
4
4
3
3
3
3
4
4
4
4
2
1
4
4
4
4
4
3
2
9.29
9.15
9.19
8.98
6.98
9.34
9.29
9.29
9.31
9.28
9.29
9.27
9.29
8.01
9.29
9.83
8.97
9.60
8.54
8.54
7.38
7.39
7.42
9.35
9.35
7.38
6.73
82.48
57.32
57.32
57.32
94.97
82.90
79.83
79.58
82.48
73.11
62.25
71.48
71.48
96.39
100.58
89.89
82.48
82.48
61.52
50.66
79.15
79.15
79.15
82.48
82.48
70.31
60.32
3.5
4.4
4.4
4.0
3.7
3.7
3.7
4.3
4.3
3.6
3.4
3.2
3.2
4.1
1.5
3.3
3.8
3.0
3.0
3.0
2.2
2.2
2.2
2.1
2.1
3.2
3.2
0.31
0.31
0.27
0.33
0.21
0.30
0.21
0.25
0.26
0.31
0.31
0.28
0.21
0.22
0.17
0.23
0.30
0.28
0.20
0.18
0.20
0.19
0.16
0.20
0.19
0.21
0.18
5.57
3.38
2.49
3.65
3.19
5.42
2.45
4.27
4.71
4.04
4.11
3.75
1.96
3.59
1.12
3.21
4.76
4.39
1.48
0.81
1.06
0.76
ꢀ0.14
1.51
1.22
2.47
0.92
9
38a
38b
38c
39
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
45a
45b
45c
46
47a
47b
47c
47d
47e
47f
47g
48a
48b
48c
48d
48e
48f
48g
48h
48i
48j
48k
a Values that are within the MPO more desired ranges are green and outside the less desired range are red. b
Calculated using ChemAxon JChem for Excel. c Calculated at pH 7.4 using ChemAxon JChem for Excel. d Calculated
using MPO calculator previously reported.14
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Table 7. ADME measurements of 1 and analogs
5
6
7
8
Rat
Aqueous
Compound Solubility
(uM)
HLM
t1/2
(min)
Rat
Hepatocyte
t1/2 (min)
Human
PPB (%) (ꢂL/min/mg)
HLM Clint
Hepatocyte
Clint
logD
7.4
(ꢂL/min/106)
9
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
6.33
298
4.02
12.2
< 1
3.77
19
> 99.9
98.61
99.8
99.8
99.9
> 99.9
*
24
22.6
15.5
12.9
34.2
43.8
80.9
142
*
*
*
24.9
81.4
17.2
29.3
33.6
49.7
98.8
33.3
7.96
30.7
17
*
4.0
2.0
4.3
4.5
4.0
4.1
*
46
*
47a
47b
47c
47d
47e
47f
47g
48a
48b
48c
48d
48e
48f
48g
48h
48j
*
*
*
*
*
*
*
*
8.6
4.9
*
7
32
*
20.8
*
*
*
87.1
*
21
99.8
99.9
> 99.9
*
36
*
*
4.3
3.7
4.0
*
4.26
11.7
*
22.5
135
*
*
*
*
61.7
> 300
100
*
*
< 2.3
*
> 99.9
> 99.9
> 99.9
> 100
100
51.6
33.9
27.3
64.2
*
*
*
*
*
*
*
*
4.9
4.3
4.7
4.0
4.4
4.9
< 1
1.39
11
*
22.5
18.7
115
*
*
0.3
< 1
*
38.8
12.8
48k
99.0
21.1
*
* Not obtained
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Table 8. Wildꢀtype and 1433ꢀresistant T. brucei growth inhibition assays. EC50s represent averages and
standard error of the mean of two independent assays.
6
7
Strain
8
9
Compound
Pentamidine
1433
WT EC50 (nM) 1433 resistant EC50 (nM) 1433 resistant EC50 / WT EC50
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1.5 ± 0.2
98 ± 1
1.1 ± 0.1
4700 ± 230
14 ± 1
0.73
48
1
0.13 ± 0.01
108
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