Improved bicyclic pyrrolidine analogs inhibit Toxoplasma gondii growth in vitro and cure infection in vivo

Abstract

Inhibition of phenylalanine tRNA synthetase (PheRS) by bicyclic pyrrolidines provides potent and specific inhibition of parasite growth. Herein we describe novel bicyclic pyrrolidines designed to explore structure-activity relationships with Toxoplasma gondii vs. human PheRS. Modification of the biaryl alkyne extension, which fits into the phenylalanine binding site, showed a strong preference for ortho hydroxyl addition over meta and para. Further addition of N to both proximal and distal phenyl rings of the biaryl alkyne and to the methoxyphenyl urea moiety, which fits into a unique auxiliary site present in the parasite enzyme, identified compounds with reduced plasma protein binding and lower hERG activity. Finally, we identified a potent lead with improved pharmacokinetics (PK), extended plasma exposure, central nervous system (CNS) penetration, and low-dose cure of acute infection in mouse. Collectively, these findings advance new candidates for treatment of toxoplasmosis based on selective and potent inhibitors of parasite PheRS.

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Introduction

Toxoplasma gondii is an opportunistic pathogen capable of causing serious disease in immunocompromised patients and during and after pregnancy due to congenital infection. It is often underappreciated as an important cause of severe ocular disease in South America^1–3. Notably, ocular toxoplasmosis in South America occurs in otherwise healthy adults and is associated with much more aggressive immunopathology^{4, 5}. Importantly, while the current standard of care using antifolates (pyrimethamine plus sulfadiazine) can control acute infection, it is not able to cure chronic infection⁶. This refractoriness to elimination is attributed to the semi-dormant status of bradyzoite stages that occur in tissue cysts in the brain, eye, and skeletal muscle⁶. The inability to cure chronic infection predisposes immunocompromised patients to reactivation and underlies the recurrent nature of ocular disease in otherwise healthy patients⁷.

tRNA synthetases play an important role in protein translation by charging tRNA with their cognate amino acids and they have been fruitful targets for identifying new classes of inhibitors that show potent and selective activity against parasites due to the different structural features of parasite enzymes vs. host⁸. Apicomplexan parasites including Plasmodium, Cryptosporidium, and Toxoplasma have a single cytoplasmic form of Phenylalanine tRNA synthetase (PheRS) that differs substantially from mammalian enzymes⁸. Previous studies have shown that specific stereoisomers of bicyclic azetidines, which contain a fused 4, 8-membered ring core, provide potent and selective inhibition of P. falciparum⁹, C. parvum¹⁰ and T. gondii¹¹, in vitro and in vivo. Structural studies with bicyclic azetidines reveal that the biaryl alkyne extension of the core fits into the phenylalanine pocket¹² and the methoxyphenyl urea extension fits into an auxiliary site where the key residues contributing to selectivity reside¹³. Among the challenges faced by this scaffold are the very high protein binding and appreciable inhibition of cardiac channels including hERG⁹.

In previous studies, we described a modified core based on a pyrrolidine (5-membered) ring fused to a diazepane (7-membered) ring, a scaffold we termed 5,7 bicyclic pyrrolidines¹⁴. This alternative 5,7 core mimics the exit-vector geometry of the 4,8 core of the active stereo configuration of parent bicyclic azetidines and thus presents the two appendages in a similar orientation¹⁴. This simplified core scaffold is also considerably easier to synthesize, allowing a greater number of analogs to be explored. Bicyclic pyrrolidines were also shown to selectively target PheRS in T. gondii vs. the host¹⁴. Although the early analogs in this series had slightly lower potency than the original 4,8 series, they presented favorable PK properties in vivo, including CNS penetration¹⁴, a desirable feature in the development of anti-toxoplasmosis therapeutics that suggested that the new chemical series warranted further exploration. In the present study, we extended the search for additional analogs of the bicyclic pyrrolidine core. We report on a lead compound that shows selective inhibition of T. gondii PheRS vs, host, potent inhibition of parasite growth in vitro, and ability to cure acute infection in a mouse model of toxoplasmosis.

Results

Previous studies have described two 5,7 bicyclic pyrrolidines that are included here for reference: 1 (previously referred to as BRD2108) which has phenyl groups in both R₁ and R₂ of the biaryl alkyne, and 2 (previously referred to as mCMY416), which has a proximal phenyl at R₂ and an cyclohexene at R₁ (Table 1)¹⁴. Although these compounds retain activity and selectivity for PheRS, they are less potent than earlier 4,8 bicyclic azetidine analogs¹⁴. To improve on the 5,7 bicyclic pyrrolidine series, we began by probing structure activity relationship (SAR) on the distal phenyl ring analogs. Introducing various ortho-substituents, including fluorine (3), methyl (4), cyano (5), to alter the electronics of the aromatic ring either had no effect or reduced the potency (Table 1). Intriguingly, a meta-phenol analog 8 enhanced the in vitro activity by over 20-fold (EC₅₀ = 0.013 μM), and potency was further enhanced in the 2-fluor-meta-phenol in compound 9. Adding N to the ring to generate the distal pyridin-ol (10) or extension of the ortho-phenyl methanol (6) offered no further improvements in potency. Further investigation of the phenol SAR reveals that the ortho-phenol, 12, offers substantial improvement and this compound exhibits nano molar potency (EC₅₀ = 0.0008 μM). In comparison, this gain in potency was not observed in aliphatic alcohol analog 7, although it has improved potency compared to the previous leads. Additionally, the catechol 13 shows much lower potency than either of the meta or ortho single substituted phenols. In contrast to the improved potency of the meta and ortho-phenols, a dramatic loss of potency was observed with the para-phenol 11 (EC₅₀ =7.69 μM). In comparison, the 5-fluoro ortho-phenol in 14 retained almost the full potency of the simple ortho-phenol 12. Collectively, the observed activity pattern suggests a role for hydroxyl positioning in modulating ligand interactions within the L-Phe binding pocket.

Table 1.

Replacement of Distal Aromatic R₁, Proximal Phenyl R₂ and Aniline R₃ SAR.

Compd No.	R1	R2	R3	AlogD	Tg EC50 (μM)	Tg EC90 (μM)	THP-1 CC50 (μM)	HEPG2 CC50 (μM)
1				4.0	0.291 ± 0.034	0.439 ± 0.043	14.5 ± 0.133	18.450 ± 0.67
2				4.5	0.202 ± 0.091	0.377 ± 0.021	12.9 ± 0.658	11.57 ± 4.39
3				4.3	0.233 ± 0.013	0.587 ± 0.17	22.67 ± 1.12	15.94±3.43
4				4.5	5.01 ± 0.525	8.21 ± 1.12	nd	nd
5				4.1	1.26 ± 0.15	1.83 ± 0.515	>20	> 20
6				3.4	1.33 ± 0.168	1.99 ± 0.557	nd	nd
7				3.7	0.0165 ± 0.0025	0.032 ± 0.016	3.95 ± 0.385	> 20
8				3.8	0.013 ± 0.002	0.043 ± 3	9.97 ± 2.93	> 20
9				4.0	0.004 ± 0.001	0.010	2.27 ± 1.06	> 20
10				4.4	0.0105 ± 0.0025	0.0335 ± 0.0005	6.42 ± 2.77	> 20
11				3.8	7.69 ± 1.11	12.42 ± 0.679	nd	nd
12				3.8	0.0008 ± 0.0002	0.0023 ± 0.0005	0.183 ± 0.115	1.12 ± 0.271
13				3.6	0.359± 0.66	0.95 ± 0.08	14.13±0.66	>20
14				4.1	0.0045±0.0025	0.0075±0.0035	0.0895 ±0.0095	2.69 ± 1.29
15				3.2	0.0026 ± 0.008	0.004 ± 0.0007	0.509 ± 0.302	4.660 ± 1.430
16				3.4	0.0016 ± 0.0017	0.0036 ± 0.0032	0.072 ± 0.031	1.69 ± 0.55
17				3.1	0.012 ± 0.0005	0.019 ± 0.005	1.48 ± 0.083	11± 2.59
18				3.0	0.092 ± 0.004	0.155 ± 0.036	4.44 ± 0.91	>20
	R 1	R 2	R 3	AlogD	Tg EC50 (μM)	Tg EC90 (μM)	THP-1 CC50 (μM)	HEPG2 CC50 (μM)
19				3.7	0.0031± 0.0012	0.0062 ± 0.0019	0.209 ± 0.08	1.18 ± 0.22
20				3.2	0.005 ± 0.001	0.011 ± 0.003	0.317 ± 0.209	1.35 ± 0.02
21				3.1	0.007 ± 0.001	0.016 ± 0.0005	2.720± 2.55	4.16 ± 0.285
22				2.7	0.025 ±0.008	0.036 ± 0.007	1.5 ± 0.377	11.8 ± 1.74
23				3.0	0.0027 ± 0.0008	0.0053 ± 0.0015	0.074 ± 0.024	14± 0.565
24				4.3	0.827 ± 0.206	1.140± 0.085	>20	>20
25				3.7	0.0165 ± 0.0005	0.061 ± 0.007	2.18±0.18	9.05 ± 0.05
26				4.1	0.0015 ± 0.0005	0.004 ± 0.000	0.005 ± 0.001	0.495 ± 0.115
27				2.7	8.528 ± 2.238	15.728 ± 6.636	nd	nd

AlogP was calculated using Schrodinger’s Canvas MolDescriptors software. AlogD was estimated from AlogP based on pH=7.4.

Next, to investigate the proximal phenyl ring SAR to improve physicochemical properties, we synthesized several heterocyclic analogs, among which pyridine replacements at C2 of the proximal phenyl ring in compounds 15 and 16 have similar activity as leading compounds 12 (Table 1). In contrast, switching of C3 to N in 17 or inclusion of a pyridazine in compound 18 led to reduced potency (Table 1).

We also attempted to replace the p-methoxyaniline, due to its known association with toxicity, with less nucleophilic amino-pyridines and other heterocycles. These derivatives were based on the favorable distal ortho-phenol identified in 12. Most derivatives showed slightly lower activity than 12, including the pyridine containing compound 19 and two pyrazine containing compounds 21 and 22, although this later compound was less potent (Table 1). Modification of the aniline to a 3-amino-pyridine was also tolerated with the proximal phenyl R2 ring containing a 2 pyridyl ring as 23 demonstrated good potency (Table 1). Importantly, all these modifications would be expected to eliminate the potential toxicity of the aniline.

Not all modifications to R3 were favorable. For example, the 2-amino pyrimidine analog 27, and addition of a 3-fluoro in compound 24 resulted in greater loss of activity against parasites (Table 1). We also explore further extensions to the para position of the aniline, since the existing crystal structures of Plasmodium PheRS suggest additional room is available in the ancillary pocket^{12, 13}. Addition of a morpholine ring in compound 20 did not compromise potency compared to the pyridine containing compound 19. Aliphatic extensions such as the fluoro-ethoxy pyridine in 25 decreased activity and capping this extension with a cyclopropyl lead to increased activity against HepG2 cells (Table 1). Although these anlogs failed to exploit differences in the anciallry binding site, future strucure guided studies may enable design of new analogs to take advanatge of this feature.

Bicyclic pyrrolidines are selective for parasite PheRS

Based on the initial assessment of potency and selectivity, we focused on a core set of eight 5,7 bicyclic pyrrolidine compounds for further analysis (Table 2). These compounds contain a range of modifications that add heteroatoms within or appended to the ring structures in R1, R2 and R3 (Scheme 1, Supplementary Experimental section; Compound Syntheses schemes). For comparison, we included the previously described 4,8 bicyclic azetidine BRD7929¹¹, as well as two previously published 5,7 bicyclic pyrrolidines 1 (BRD2108) and 2 (mCMY416)¹⁴(Table 2). Initially, we tested the compounds for on-target activity based on known mutations in PheRS that render the enzyme resistant to inhibition by the 4,8 series of inhibitor¹¹ as well as early leads of the 5,7 series¹⁴. We used the point mutation L497I in PheRS in the type I RH strain to test the top compounds in the 5,7 bicyclic pyrrolidine series. Although the RH strain grows faster in vitro, previous studies indicate that it has comparable sensitivity to the bicyclic pyrrolidines 1 and 2 when compared to the type II ME49 strain used for the determination of EC₅₀/EC₉₀ values (Table 2)¹⁴. Among the most active analogs, the fold resistance in comparing EC₅₀ growth inhibition of the mutant to the parental RHΔΔ parasite ranged from ~ 10 to > 300-fold (Table 2). Of note, 12 was intermediate in this range with a resistance change of ~ 50-fold. We also tested the compounds for growth inhibition of mammalian hepatic HepG2 cells and differentiated mononuclear phagocyte THP-1 cells (Table 2). HepG2 cells proved to be insensitive to the compounds and comparing the CC₅₀ of host cell proliferation to EC₅₀ of parasite growth revealed selectivity indexes (SI) ranging from 35 to > 2,000 (Table 2). THP-1 cells were somewhat more sensitive to growth inhibition but still showed SI that ranged from ~20 to > 400 (Table 2). These findings are consistent with the compounds being selective inhibitors of TgPheRS, as further supported below.

Table 2:

Potency and selectivity of top active candidates

Compd No.	TgM49a EC50	TgME49a EC90	TgL497I EC50/ Parentalb EC50 (FR)	Tg68c EC50 (Alkaline)	Tg68c EC50 (Glutamine)	HepG2 CC50	SI	THP-1 CC50	SSII
BRD7929	0.041	0.139	78	0.201	0.070	6.07	147	1.01	24
1	0.305	0.441	10	0.340	0.169	15.44	51	14.54	48
2	0.331	0.524	8	0.938	0.218	11.57	35	12.93	39
7	0.037	0.064	343	0.005	0.010	19.69	539	8.04	220
8	0.022	0.043	78	0.023	0.014	17.72	810	9.97	455
10	0.019	0.040	100	0.005	0.011	38.70	2056	7.24	385
12	0.001	0.002	47	0.003	0.0001	0.99	1447	0.25	374
15	0.003	0.004	36	0.002	0.001	3.80	1462	0.51	196
16	0.004	0.008	22	0.001	0.00003	1.42	349	0.07	18
17	0.007	0.012	38	0.012	0.008	9.08	1239	1.48	202
19	0.003	0.005	50	0.003	nd	1.06	369	0.21	73
20	0.004	0.007	61	0.001	0.003	1.31	313	0.32	75
21	0.007	0.015	28	0.077	nd	5.39	797	2.72	402
22	0.003	0.023	314	0.004	0.005	10.06	2981	1.50	443
23	0.003	0.004	20	0.004	0.005	1.56	487	0.07	23
Atovaquone	0.071	0.163	nd	1.592	0.052	nd	nd	nd	nd
Pyrimethamine	0.330	0.551	nd	nd	nd	nd	nd	nd	nd

Values in μM, average of three or more biological replicates.

^aME49 expressing firefly luciferase,

^bParental strain used is TgRHΔhxgprtΔku80,

^cTg68 nLuc expressing Nano luciferase

FR = EC₅₀ fold resistance between BRD7929 resistant parasite line TgL497I and parental parasite line TgRHΔΔ.

SI = selectivity index (Host cell CC₅₀ / EC₅₀ TgME49).

nd = not determined.

Correlation of growth inhibition and enzyme selectivity

To further support the specificity of the inhibitors for PheRS, we expressed the parasite (TgcPheRS) and host (HscPheRS) enzymes in E. coli (Figure S1). Representative 5,7 bicyclic pyrrolidine compounds were evaluated for inhibition of enzyme activity in vitro. Initially, we tested them using a thermal shift melting assay where tighter compound binding to the active site results in a greater difference in the temperature of melting (ΔTm). Incubation of the TgcPheRS enzyme with inhibitors led to ΔTm values ranging from 10–14 °C, while this value was typically < 6 °C for the human enzyme (Table S1). We observed an iinverse correlation (R² = 0.79) between the ΔTm of compounds with TgcPheRS and their potency in inhibiting parasite growth in vitro (Figure 1A, Table S1). Notably, because all of the compounds examined here are inhibitors of TgcPheRS, the range of ΔTm values from highly potent to moderately potent compounds only changes slightly. Nonetheless, even these modest drops in ΔTm correlate with appreciable loss of potency against the parasite enzyme (Figure 1). We also observed a positive correlation (R² = 0.61) between the EC₅₀ based on parasite growth inhibition vs. IC₅₀ against parasite enzyme activity (Figure 1B, Table S1). In contrast, IC₅₀ values for inhibition of the host enzyme were 50–100 times higher (Table S1). Structure-based sequence alignment between TgcPheRS and HscPheRS revealed four non-conserved residues within the 5Å radius of mCOI226 consisting of these changes: TgV411/HsI373, TgY433/HsF395, TgI436/HsL398 and TgI468/HsV430). These four variable residues likely mediate selectivity for the parasite enzyme over human, as previously reported for Plasmodium falciparum (PfcPheRS) ⁽¹²⁾. These findings further support the conclusion that the 5,7 compounds are potent and selective inhibitors of the parasite PheRS enzyme and that it is the primary target underlying parasite growth inhibition in vitro.

Figure 1.

Correlation of enzyme inhibition and parasite growth inhibition. A) comparison of change in thermal meting (ΔTm) for parasite PheRS vs. EC₅₀ for parasite growth inhibition. B) Comparison of parasite PheRS enzyme inhibition (IC₅₀) to parasite growth inhibition (EC₅₀). Points represent means from N =3 experiments. See Table S1 for individual values.

The majority of bicyclic pyrrolidines are fast-acting and irreversible

To determine if the 5,7 bicyclic pyrrolidine series compounds act rapidly or if they require a prolonged incubation for inhibition, we compared treatment for 4 h followed by washout and re-culture in the absence of compound to continuous treatment for 72 h. Most of the bicyclic pyrrolidines were highly potent even when used for only 4 h and showed comparable EC₅₀ values to continuous treatment (Figure 2A). Notably, compound 12, which is featured below, show equal potency when treated or 4 h vs continuously. Notable exceptions included compounds 7, 10, 15, and 22 that showed > 10-fold increase in EC₅₀ values when only treated for 4 vs. 72 h (Figure 2A). The reason some compounds required extended treatments is not evident from comparing either their relative potencies (Table 2), or predicted hydrophobicity (Table 1), but may result from differences in uptake and retention in cells. In contrast to the rapid action of most bicyclic pyrrolidines, atovaquone, which targets the mitochondrial bc1 complex¹⁵, and pyrimethamine, which inhibits DHFR^{16, 17}, were only effective when used for 72 h treatment (Figure 2A).

Figure 2.

Time dependent inhibition of tachyzoite and bradyzoite growth of T. gondii by bicyclic pyrrolidines. A) Time dependent inhibition of T. gondii tachyzoites with top analogs. Points indicate 4 vs. 72 h EC₅₀ values from two or more experiments (mean ± S.D.). B) Time dependent inhibition of ex vivo purified bradyzoites. One-way ANOVA with multiple comparisons of column means to the DMSO control from two or more experiments (mean ± S.D. * P < 0.05), with variance homogeneity assessed by Brown-Forsythe and Bartlett’s tests. C) Time dependent inhibition of bradyzoites within intact cysts. Top panels show schematic for experimental design. Bottom panels indicate means from two or more experiments (mean ± S.D.), N= 2,3 experiments shown as individual data points). One-way ANOVA with multiple comparisons of column means to the DMSO control, * P < 0.05, with variance homogeneity assessed by Brown-Forsythe and Bartlett’s tests.

Bicyclic pyrrolidines act on in vitro differentiated and ex vivo derived bradyzoites.

Although the bicyclic pyrrolidines showed potent activity against tachyzoite stages of T. gondii in vitro, bradyzoites are thought to be much harder to inhibit due to their semi-dormant state. We tested the top active bicyclic pyrrolidines against in vitro differentiated bradyzoites using two methods for differentiation including high pH shock and reduced glucose supplemented with glutamine, as recently described¹⁸. For these assays, we used a Nano luciferase expressing strain called Tg68 (Tg68nLuc), which is permissive for bradyzoite induction in vitro¹⁹. The bicyclic pyrrolidines performed well against bradyzoites in both assays with EC₅₀ values that were comparable in most cases including 12 and elevated by 3 to 5-fold in other cases (e.g. BRD7929, 2), when compared to tachyzoite conditions. (Table 2). Several compounds showed enhanced activity against bradyzoites induced using glutamine-rich media, including 12 and 16 (Table 2).

We also tested the top active compounds against bradyzoites purified from tissue cysts isolated from the brains of chronically infected mice. Initially, we tested the effects of compounds on bradyzoites after trypsin-mediated digestion of the cyst wall. Compounds were added at 3X EC₉₀ for 4 h and then washed out and re-cultured on HFF monolayers without compound vs. continual treatment during outgrowth, during which the parasite converts to tachyzoites and forms plaques in the monolayer (Figure 2B). Most compounds acted rapidly and only 4 h treatment was necessary to observe a substantial reduction in subsequent outgrowth (Figure 2B). However, a subset of compounds, including several that showed slower inhibition kinetics on tachyzoites in vitro (e.g. 7, 15, and 23), were only active when used continuously (Figure 2B). Consistent with the results on tachyzoite growth inhibition, atovaquone and pyrimethamine were only active when used continuously (Figure 2B).

We used a similar protocol to test the effect of bicyclic pyrrolidines on intact cysts purified from the brains of chronically infected mice. In this assay, compounds were added at 3X EC₉₀ to intact cysts for 4 h or 24 h then removed by centrifugation, three times wash with PBS and medium replacement. Bradyzoites were then liberated by trypsin treatment and plated on HFF monolayers in the absence (washout) vs. presence (constant) of compounds (Figure 3C, S2). None of the compounds was able to inhibit outgrowth after only 4 h of treatment (Figure S2); however, several of the compounds showed up to 50% inhibition when used for 24 h of treatment followed by washing and plating in the absence of compounds (e.g. BRD7929, 1, 2, 10, 16, 17) (Figure 2C). The properties that determine these differences are not evident from examining the lipophilicity of the molecules (Table 1) nor their potency (Table 2) or time-dependent killing ability on tachyzoites or liberated bradyzoites (Figure 2A, B). The differential activity on intact cysts may reflect their ability to cross the cyst wall, which is comprised of a dense meshwork of glycoproteins with poorly defined permeability^{20, 21}. Notably, atovaquone, which has previously been shown to reduce the burden of cysts in chronically infected mice²², and delay the reactivation of chronic CNS infection in immunocompromised mice²³, was among the most effective compounds in this assay (Figure 2C). Although the properties that determine the uptake and activity of compounds on intact cysts are unknown at present, this assay provides a useful surrogate for how compounds might act in vivo.

Figure 3. PK profile of 12.

A) Structure of compound, B) Plasma levels of 12 were measured after single (QD) oral doses at 0.5, 1, 3, 8, and 24 hours (n = 3 per group), with values as means ± S.D. based on unbound plasma fractions. C) Plasma levels of 12 after 10h apart double (BID) oral dose of compound for 1 day measured at 1, 3, 8, 24 h (n = 3 per group), with values as means ± S.D. based on unbound plasma fractions. D) Plasma levels of 12 after 10h apart double (BID) oral dose of compound for 4 days measured at 1, 3, 8, 24 h (n = 3 per group), with values as means ± S.D. based on unbound plasma fractions. Compound suspended in 75% PEG 300, 25% DSW. n=3 animals per time point. 10X EC₉₀ estimated from in vitro activity.

ADME properties of top active compounds

To further evaluate the top 5,7 bicyclic pyrrolidine compounds, we profiled them for in silico physicochemical properties as well as several in vitro assays designed to estimate their ADME properties (Table 3). As indicated by computational analyses, most compounds were highly hydrophobic, like the previously described compounds used here for reference including, BRD7929 (ALogD = 4.4), 1 (AlogD = 4.0), and 2 (AlogD = 4.5). However, some improvement was seen with the new analogs, especially with 22 (AlogD = 2.7), which contains an ortho hydroxyl at R1 and a methoxypyrazine in the R3 moiety (Table 3). The reference compounds showed high plasma protein and brain homogenate binding, and this property was shared by the new analogs, although we only tested a few representative ones (Table 3). The reference compound BRD7929 showed excellent stability in mouse liver microsomes, while compounds 1 and 2 showed moderate stability (Table 3). However, phenol analogs (e.g. 12 and 16) and cyclohexyl alcohol analog 7 show lower stability in mouse liver microsomes in vitro, likely due to the hydroxyl moiety (Table 3). Low absorption (permeability A-B) and high levels of efflux (permeability B-A) were generally found for most compounds, except for the previously described 2 (Table 3). Some improvement in the uptake rate (permeability A-B) was seen with 22, which contains an ortho hydroxyl at R1 and a methoxypyrazine in the R3 moiety (Table 3). Finally, hERG activity has previously been reported with 4,8 bicyclic azetidine compounds such as BRD7929⁹. Relatively high inhibition of hERG was also observed for the new compounds, although improvement was seen in several compounds including 12 (hERG inhibition 0.83 μM), which as a distal orthro hydroxyl at R1, to 19 (hERG = 2.33 μM), which shares the ortho hydroxyl at R1 and contains an ethoxy pyridyl at R3, and 20 (hERG = 2.51 μM), which has the orthro hydroxyl at R1 and a pyridyl, morpholine at R3 (Table 3). Collectively, these trends suggests that further modifications to the saturated rings of the 5,7 bicyclic pyrrolidine core and methoxyphenyl urea moiety may further reduce hydrophobicity, decrease efflux, and reduce inhibition of hERG.

Table 3.

ADME profiles for top compounds.

	AlogD	AlogP	Tg EC90 (uM)	Plasma protein binding % (CD-1 mouse)	Plasma protein binding % (human)	Brain Homogenate binding %(CD-1 mouse)	Microsomal stability mouse (μL/min/mg)/Eh	hERG inhibition CHO cell IC50 (μM)	MDCK-MDR1 Permeability A-B Papp(10−6cm/s)	MDCK-MDR1 Permeability B-A Papp(10−6cm/s)
BRD7929	4.4	6.4	0.134 ± 0.085	99.6	99.56	99.98	15.41/0.40	0.54	0.28	9.62
1	4.0	6.1	0.439±0.043	>99.9	nd	nd	35.82/0.61	0.23	0.45	1.28
2	4.5	6.5	0.377±0.021	>99.9	99.8	99.99	49.8/0.69	1.97	0.02	0.66
7	3.7	5.6	0.032±0.016	98.8	nd	nd	147.91/0.87	nd	0.87	64.19
8	3.8	5.8	0.043 ± 0.003	nd	nd	nd	nd	nd	0.47	12.38
10	4.4	4.7	0.0335 ± 0.0005	nd	nd	nd	nd	nd	nd	nd
12	3.8	5.8	0.0023±0.0005	99.9	99.4	99.9	137.8/0.86	0.83	0.61	11.64
15	3.2	5.1	0.004±0.0007	nd	nd	nd	nd	0.2	0.65	40.47
16	3.4	5.3	0.0036±0.0032	nd	nd	nd	73.59/0.76	0.56	nd	nd
17	3.1	5.0	0.019±0.005	nd	nd	nd	nd	0.11	2.2	36.86
19	3.7	5.6	0.0062±0.0019	nd	99.58	nd	nd	2.33	0.38	15.04
20	3.2	5.1	0.011±0.003	nd	99.24	nd	nd	2.51	0.92	16.33
21	3.1	5.0	0.016±0.0005	nd	nd	nd	nd	0.36	nd	nd
22	2.7	4.6	0.036±0.007	99.6	nd	99.7	nd	0.8	3.01	19.25
23	3.0	4.9	0.0053±0.0015	nd	99.1	nd	nd	nd	0.06	58.47

nd = not determined. Eh, Hepatic extraction calculated mouse liver blood flow of 90ml/min/kg. See also Figure S3

Pharmacodynamic (PK) studies

To evaluate the pharmacokinetic properties of the compound in vivo, we monitored plasma and brain exposure after dosing per oral with compounds in several different vehicles designed to ensure a uniform suspension (Table 4). Previous compounds such as BRD7929, 1, and 2 showed good C_max exposure when adjusted for dose, modest AUC_last adjusted for dose, and good brain exposure (Table 4). However, we have previously encountered issues with increased exposure and toxicity with repeated dosing of BRD7929¹¹, likely due to its high tissue distribution (e.g. Vss ~30 l/kg ⁹). Also, the compounds 1, and 2 were less potent against the parasite in vitro (Table 2) and hence the exposure achieved in vivo would be unlikely to reach sufficient levels for efficacy. Hence, we explored the PK of selected new compounds, particularly 12 which shows very potent inhibition of the parasite in vitro (Table 1). Compound 12 showed modest oral bioavailability of ~ 10%, relatively low C_max, and modest AUC_last values (Table 4). When dosed once a day (QD) compound 12 showed good exposure in plasma and at 30 mg/kg. When dosed QD, compound 12 exceeded the 10X EC₉₀ total levels in plasma for 24 hr (Figure 3), while exposure dropped below this value by ~15 hr for 10 mg/kg and by 8 hr for 3 mg/kg (Figure 3). When doses twice a day (BID) compound 12 showed good plasma exposure that rose gradually over 8 hr although it fell below 10X EC₉₀ by 24 hr with both a single day of dosing and after 4 days of dosing (Figure 3 C,D) The initial increases during the first 4–6 hr are likely due to gradual oral absorption combined with intrinsic clearance rates. Notably, 12 had a prolonged half-life T_½ of 10–15 h (Table 4). The brain-to-plasma ratio of 12 was 0.884 at 8 h following 10 mg/kg oral dosing. In contrast, the meta phenol at R1 found in 8 had very poor absorption and was barely detectable in plasma. Among the other analogs, none showed marked improvement in any of the PK parameters and most failed to achieve extended plasma coverage above 10X EC₉₀ (e.g. 8, 10, 20) (Table 4). Compounds 15, 16, and 19, exhibited good plasma exposure but had poor CNS penetration with low brain / plasma ratios (Table 4, Table S2, Figure S3).

Table 4.

Comparison of pharmacokinetic parameters for top compounds.

	Dose(mg/kg)	Formulation	Plasma Cmax/D (ng/mL)	Plasma tmax (h)	Plasma t1/2 (h)	Plasma AUClast/D (hr*ng/mL)	Oral F%	Brain Cmax/D (ng/mL)	Brain tmax (h)	Brain AUClast/D (hr*ng/mL)	Mouse B/P (time)
BRD7929	10 (PO)	A	40.6 ± 10.4	8	na	220.9	ND	179 ± 60.7	8	807	4.4 (8h)
1	10 (PO)	A	32.5 ± 5	3	na	195.1	ND	304 ± 132	8	2121	9.13 (3h)
2	10 (PO)	A	11.2 ± 2.2	1	na	26.8	ND	33.9 ± 7.6	1	108.1	3.03 (1h)
7	10(PO)	A	44.8 ± 22.2	3	na	193	nd	5.42 ± 0.1	1	24.3	0.26 (8h)
8	10 (PO)	A	0.5 ± 0.2	1	na	1.3	nd	na	na	na	na
9	10 (PO)	A	na	nd	na	na	nd	na	nd	na	na
10	10 (PO)	B	0.6 ± 0.7	1	na	1.3	nd	na	nd	na	na
12	2 (IV)	B	209 ± 68	0.5	3.73	482	-	nd	nd	nd	nd
12	10 (PO)	B	10.4 ± 5.3	1	3.54	45.1	9.4	nd	nd	nd	nd
12	3 (PO)	A	12.8 ± 0.81	0.5	15.5	43.3	9	nd	nd	nd	nd
12	10 (PO)	A	5.6 ± 6.3	3	nd	25.8	5.3	28.3 ± 16.2	1h	16.8	1.58 (8h)
12	30 (PO)	A	10.6 ± 1.32	0.5	10.5	69.2	14.3	nd	nd	nd	nd
15	100 (PO)	B	43.9 ± 15.7	1	1.37	93.6	nd	nd	nd	nd	0.154 (8h)a
16	10 (PO)	A	3.66 ± 1.09	1	5.4	21	nd	na	nd	na	0.005 (1h)
19	10 (PO)	B	13.4 ± 9.1	1	na	34.8	nd	1.45 ± 0.12	3	9.8	0.614 (8h)
20	10 (PO)	A	2.75 ± 0.2	3	na	16.8	nd	nd	nd	na	na

Formulation: A solution −75% PEG300, 25% D5W; B solution-20% PEG 400, 10% Vitamin E TPGS, 70% sodium acetate buffer (50mM, pH = 4.1); 3-0.2% MC, 0.2%Tween80

nd: not determined. na: not detected

^a-only terminal b/p available at 8h

Compound 12 provides protection against infection in vivo

Based on its more favorable ADME and PK properties that allow extend exposure in plasma compared to compounds 1,2, 7 and 16 that showed less stability in microsomes and/or lower plasma exposure over time. we chose compound 12 to more forward with. We tested compound 12 in vivo for efficacy against toxoplasmosis in the immunocompetent murine model using 6–8 week old C57/BL6 female mice. We used the ME49 strain, which has intermediate virulence in the mouse, and injected 300 tachyzoites i.p. to simulate an LD₅₀ dose (Figure 4A). We waited 4 days to allow the infection to establish and then began treatment by oral gavage of compounds given daily for 7 days. Based on the PK properties described above, we choose doses of 10 mg/kg QD and 3 mg/kg BID for 12. In addition, we included a group given the previous 4,8 compound BRD7929 at 10 mg/kg QD, and a group given sulfadiazine in the drinking water at 0.25 mg/ml (equals an average daily dose of ~1.5 mg / animal). As expected, we observed ~ 50% death in the vehicle control group, although the time to death was delayed, suggesting a lower level of infection than anticipated (Figure 4B). Vehicle control mice also experienced high levels of weight loss, which began to increase in surviving animals after 3 weeks (Figure 4C). In comparison, mice given sulfadiazine, BRD7929 and 3 mg/kg BID 12 experience little weight loss and minimal mortality (Figure 4B,C). Animals given 12 at 10 mg/kg QD experienced considerable weight loss during the period of treatment, suggesting this high dose was toxic (Figure 4C).

Figure 4. In vivo efficacy studies with 12 in immunocompetent mice.

A) Schematic for experiment 1. C57/BL6 mice (8/group) were infected via i.p. injection with 300 TgME49-FLuc tachyzoites, treated from 4 to 10 DPI, and monitored for 30 days. Infection in survivors was confirmed by serum ELISA, and in vitro culture, and plaque assay of brain homogenate. B) Survival curves for experiment 1, monitored for 30 DPI. Mice treated p.o. with 12: 3 mg/kg BID (10 h apart), 12: 10 mg/kg QD, BRD7929: 10 mg/kg, Sulfadiazine 0.25 mg/ml in water or Vehicle control. Mice except 3 mg/kg BID group survived significantly longer than vehicle control mice (*, P <0.05, Mantel-Cox test). All surviving mice were positive for T. gondii infection by ELISA and verified to be cured by outgrowth on HFF monolayers (Fig. S4A, Table 5). C) Weight loss for experiment 1. Vehicle control mice experienced significant weight loss, whereas mice treated with sulfadiazine, BRD7929, and 3 mg/kg BID 12 showed minimal weight loss (*, P < 0.05, two-way ANOVA, Tukey test). D) Schematic for experiment 2, C57/BL6 mice (8/group) were infected via i.p. injection with 1000 TgME49-FLuc tachyzoites, treated from 4 to 10 days post-infection (DPI), and monitored for 30 days. Survivors’ infection was confirmed by serum ELISA, and in vitro culture, and plaque assay of brain homogenate. E) Survival curves for experiment 2, monitored for 30 DPI. Mice treated with 12: 1 mg/kg, 12: 3 mg/kg, or Vehicle control, BID (10 h apart). Mice survived significantly longer than vehicle control mice (*, P <0.05, Mantel-Cox test). All surviving mice were positive for T. gondii infection by ELISA and verified to be cured by outgrowth on HFF monolayers (Fig. S4B, Table 5). F) Weight loss for experiment 2. Vehicle control and 12: 1 mg/kg mice experienced significant weight loss, whereas mice treated with 3 mg/kg BID 12 showed minimal weight loss (*, P < 0.05, two-way ANOVA, Tukey test). DPI, days post infection, p.o., per oral, ip, intraperitoneal, Rx, treatment.

Given the high rate of survival in this experiment, we also examined the animals by ELISA performed on serum collected at ~ 30 days post-infection²⁴. All the animals were seropositive, indicating they did become infected (Figure S4A). We also isolated the brain from surviving animals and examined it for the presence of tissue cysts, using standard protocols for staining with Dolichos biflorus lectin²⁴. Consistent with the lower intensity of infection, we did not detect tissue cysts in brain homogenates from any of the animals, although the sensitivity of this assay is limited to > 50 cysts / brain. To determine if the surviving mice harbored chronic cyst stages, we also inoculated mouse brain homogenates onto monolayers of HFF cells in 6 well plates to performing plaquing assays and into T25 flasks containing HFF cells to isolate any surviving parasites. These results indicate that all 4 mice surviving in the vehicle control group harbored chronic infection (Table 5), as well as 2 of 8 mice given 12 at 10 mg/kg QD, 1 of 8 surviving mice given BRD7929 at 10 mg/kg QD, and 7 of 8 mice given sulfadiazine (Table 5). In contrast, none of the mice given 12 at 3 mg/ml BID showed evidence of chronic infection (Table 5).

Table 5.

Outcome of efficacy treatment as established by outgrowth of brain homogenate samples from surviving mice

Experiment 1	Experiment 2	Survivors	Plaque positivea	Culture positiveb	EC50c
Vehicle Control : BID (n=8)		4	4	4	nd
12 : 3 mg/kg BID (n=8)		7	0	0	nd
12 : 10 mg/kg QD (n=8)		8	2	2	No change
BRD7929 : 10 mg/kg QD (n=8)		8	1	1	No change
Sulfadiazine 0.25 mg/ml (n=8)		8	7	6	nd
	Vehicle Control : BID (n=8)	2	2	2	nd
	12 : 1 mg/kg BID (n=8)	7	2	7	No change
	12 : 3 mg/kg BID (n=8)	8	0	0	nd

^aPositive in plaquing assay after 14 days

^bPositive by outgrowth in HFF culture

^cEC₅₀ determination using TgME49 expressing firefly luciferase

nd: not determined

Based on the outcome of the initial experiment, we repeated the efficacy test with several changes. First, we increased the inoculum of parasites to 1,000 tachyzoites i.p. to assure greater intensity of infection (Figure 4D). Second, we discontinued testing at 10 mg/kg out of concern for possible toxicity. Instead, we compared doses of 12 at 1 mg/kg and 3 mg/kg BID given over a similar time frame from day 4 to 11 postinfection (Figure 4D). This second experiment led to greater mortality in the vehicle control group while only a single animal succumbed at 1 mg/kg BID 12. Unlike the other two groups, animals given 3 mg/kg BID 12 did not lose weight and all animals survived infection (Figure 4E,F). All the surviving animals were seropositive, indicating they all initially got infected (Figure S4B). Comparison of the outgrowth of parasites from brain homogenates indicated that all surviving mice given sulfadiazine or 12 at 1 mg/kg BID harbored chronic infection (Table 5). However, at a dose of 3 mg/kg 12 animals showed no sign of residual chronic infection (Table 5). We also examined plasma samples from mice treated in parallel with similar doses of 12 for evidence of liver or kidney toxicity using serval commonly sampled enzymes (Table 6). We did not observe any significant increase in any of the indicator enzymes (Table 6), suggesting a lack of toxicity at the doses of 12 used in the efficacy studies. Collectively, these findings indicate that at modestly low doses, 12 protects against lethal infection and prevents establishment of chronic infection in immunocompetent animals.

Table 6.

Analysis of plasma enzyme levels in mice treated with 12.

Analyte	Vehicle Control: BID n=7	12 : 1 mg/kg BID n=8	12 : 3 mg/kg BID n=8
ALT (U/L)a	20.14 ± 1.95	20.75 ± 1.98	22.25 ± 3.01
AST (U/L)b	49.43 ± 10.37	48.88 ± 9.52	62.25 ± 21.09
BUN (mg/dL)c	20.86 ± 1.77	21.13 ± 1.81	19.13 ± 3.27
Creatinine (mg/dL)	0.31 ± 0.03	0.33 ± 0.02	0.33 ± 0.05
Total Protein (g/dL)	5.73 ± 0.33	5.69 ± 0.15	5.59 ± 0.38
Glucose (mg/dL)d	124.57 ±3 3.17	146.63 ± 17.05	142.88 ± 29.02

Values represent mean ± S.D.

^aAspartate amino transferase

^bAlanine amino transferase

^cBlood urea nitrogen

^dnon-fasting

In addition, we tested compound 12 for efficacy against toxoplasmosis in a immunodeficient murine model using 6–8 week old interferon gamma receptor 1 knockout mice (Ifngr1^−/−) mice. Mice were infected with 300 ME49 tachyzoites i.p. and treated by oral gavage of compounds daily from 1–14 days post infection (Figure 5A). Based on the PK properties described above, we choose doses of 10 mg/kg QD and 3 mg/kg BID. In addition, we included a group given sulfadiazine in the drinking water at 0.25 mg/ml (equals an average daily dose of ~1.5 mg / animal). As expected, we observed ~ 50% death in the vehicle control group by 8 days post infection, and 100% by 14 days post infection (Figure 5B). In comparison, mice given sulfadiazine, 3 mg/kg BID 12 survived significantly longer (Figure 5B).

Figure 5. In vivo efficacy studies with 12 in immunodeficient mice.

A) Schematic for experiment. Interferon gamma receptor 1 knockout (Ifngr1^−/−) mice (5– 11/ group) were infected via i.p. injection with 300 TgME49-FLuc tachyzoites, treated from 1 to 14 DPI, and monitored for 30 days for survival and body weight loss. B) Survival curves. Mice treated p.o. with 12: 3 mg/kg BID (10 h apart, n=11), 12: 10 mg/kg QD (n=5), Sulfadiazine 0.25 mg/ml in water (n=11) or Vehicle control (n=11). Mice except 12: 10 mg/kg QD group survived significantly longer than vehicle control mice (*, P <0.05, Mantel-Cox test). 12: 3 mg/kg BID mice survived significantly longer than sulfadiazine group mice (*, P <0.05, Mantel-Cox test). C) Weight loss. Mice treated with sulfadiazine showed minimal weight loss earlier (DPI 2–4, *, P < 0.05, two-way ANOVA, Tukey test). DPI, days post infection, po, per oral, ip, intraperitoneal, Rx, treatment.

Discussion and Conclusions

We have identified, via structure-activity relationship studies on a recently reported 5,7 bicyclic pyrrolidine series of T. gondii PheRS inhibitors, analogs that feature increased in vitro antiparasitic potency in addition to reduced hydrophobicity and toxicity, and increased coverage over EC₉₀ in mice plasma. We further show that these series analogs inhibit the parasite enzyme with selectivity over the human ortholog. In particular, lead compound 12 shows promise for the development of new therapeutic agents to treat acute infection with T. gondii. Compound 12 contains an ortho hydroxyl in the distal ring of the biaryl acetylene extension from the core. The placement of this hydroxyl proved to be crucial to in vitro antiparasitic activity as the meta and para isomers were far less active and altogether inactive, respectively. The basis for this difference in potency would not be predicted from physicochemical properties and is presently under investigation through X-ray crystallization studies. In the meantime, speculate that the ortho hydroxyl establishes favorable interactions within the L-Phe binding site of the target enzyme. The pharmacokinetic profile of this lead compound suggests modest bioavailability, Cmax, and T_1/2 that provides extended coverage in both plasma and brain and yet allows clearance over time. Compared to our previous lead, the 4,8 bicyclic azetidine BRD7929, the profile of 12 is predicted to afford greater safety as it is unlikely to accumulate based on 4-day repeated dosing. In contrast, the previous lead 7929 has a very long half-life and high volume of tissue distribution (Vss ~30 at day 4) resulting in tissue accumulation with repeated dosing^{9, 11}. Although 12 does show some liability in inhibition of hERG, its potent activity against the parasite may overcome this deficiency as it was effective in vivo at very low oral dosing. Finally, 12 performed better than both BRD7929 and sulfadiazine in preventing lethal acute infection in immunocompetent mice. However, treatment of immunodeficient mice with 12 only resulted in modest extension of survival in immunodeficient mice, consistent with the finding that it was not highly effective against intact tissue cysts.

Identifying compounds with better CNS penetration and potency against bradyzoites within intact tissue cysts will be important to improve performance in chronic infection models and to advance future preclinical candidates. Additionally, there are remaining issues with the bicyclic pyrrolidine scaffold that include modest hERG activity, high protein binding, and elevated efflux for some compounds. We also observed that at higher doses, 12 led to weight loss, although the basis of this toxic effect is unknown. These remaining issues could be potentially addressed through synthesis of additional analogs and through structure-guided design as there is ample room to expand the chemical space relative to the binding interaction with the target PheRS enzyme.

Experimental section

Compounds synthetic schemes and NMR spectra of the final compounds are provided in the Supplementary Experimental section. All compounds tested in the biological assays were >95% pure by ¹H NMR. The majority of compounds were analyzed by low resolution mass spectrometry, expect for 12 for which high resolution MS data were obtained, and traces are found in the Supporting information.

In vitro Growth Inhibition and Cytotoxicity assays

Compounds were obtained from Calibr (Scripps Research, La Jolla, CA 92037, USA) as lyophilized powder or 10 mM stocks in 100% DMSO and stored at −80°C. Human foreskin fibroblasts (HFF) and parasite cultures were cultured in a medium consisting of 10% DMEM (Dulbecco’s Modified Eagle Medium), supplemented with 10 mM glutamine, 10 μg/mL gentamycin, and 10% fetal bovine serum. The cultures were incubated at 37°C with 5% CO₂ and routinely confirmed negative for mycoplasma contamination using the e-Myco Plus kit from Intron Biotechnology.

Luciferase-based growth assays were conducted using the previously described TgME49-Fluc strain²⁵. To reduce variability, only the inner 60 wells of white 96-well plates (Costar) were used to generate a 3-fold series of compounds. Each well received 5×10³ freshly harvested tachyzoites cultured in confluent HFF in 100 μL volumes, achieving a final compound concentration (200 μL/well, 0.1% DMSO in 10% DMEM). Plates were incubated for 72 h at 37°C before analysis, following the Promega Luciferase Assay System protocol. In a similar setup, plates were incubated for 4 h at 37°C, washed three times with PBS, and then incubated for an additional 68 h at 37°C. After incubation, culture media were replaced with 50 μL of 1x Cell Culture Lysis Reagent (Promega). After 10 min at room temperature, 100 μL of Luciferase Assay reagent was added, and luciferase activity was measured using a BioTek Cytation 3 multi-mode imager with Gen5 software.

To evaluate the on-target mechanism of inhibitors, compounds were tested against a resistant strain of T. gondii named TgRH-PheRS^[L497I]-FLuc that was generated in the parental TgRHΔΔ-FLuc (type I) strain, described previously¹¹. To test inhibitors on bradyzoites, a permissive bradyzoite-inducible strain of T. gondii, Tg68pBAG1:nLuc,DHFR (type II) was used, as previously documented^{18, 19}. Freshly harvested Tg68pBAG1:nLuc,DHFR parasites (3×10^3) in a 100 μL volume were added to the inner 60 wells of a black-bottom, black 96-well plates (Costar) containing monolayers of HFF cells and incubated for 2 h at 37°C in a 5% CO₂ incubator to allow parasite invasion. Afterwards, the culture medium was replaced with either 200 μL/well of alkaline medium (RPMI 1640 containing 1% FBS and 50 mM HEPES, adjusted to pH 8.2) or glutamine medium (glucose-free RPMI 1640 containing 1% FBS, 50 mM HEPES, and 10 mM glutamine, adjusted to pH 7.2). The cultures were incubated at 37°C in ambient CO₂ and maintained for 10 days, with media changes on days 3 and 6. On day 6, the plates were aspirated, and 150 μL/well of fresh media was added, followed by 50 μL/well of a dilution series containing 4X concentrations of compound. On day 10, the plates were equilibrated to room temperature for 30 min prior to the assay readout. The medium was aspirated, 50 μL of Promega Nano-Glo reagent was added to each well, and the plates were covered with a black lid. After 10 min at room temperature the reaction was read using a BioTek Cytation 3 equipped with Gen5 software.

Human hepatocellular carcinoma cells (HepG2, ATCC-HB-8065) and the human monocytic tumor line (THP-1, ATCC-TIB-202) were used to assess toxicity against host cells, as described previously¹⁸. For toxicity screening, compounds were diluted to 120 μM (2x concentration in 10% DMEM, 0.4% DMSO) and then serially diluted in a 10-dose, 3-fold series in 10% DMEM. Host cells were seeded at a density of 10×10³ cells/well in 100 μL volumes, allowing sub-confluent monolayers to expand during the 72 h growth assay. THP-1 cells were treated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 24 h to differentiate into macrophages before compound addition. For HepG2 cells, compounds were added 6 h post-seeding into plates containing host cells (200 μL final volume, 0.2% DMSO) and incubated at 37°C with 5% CO₂. After 72 h, the culture media were aspirated to 80 μL, an equal volume of CellTiter-Glo^® Luminescent Cell Viability Assay reagent (Promega) was added, and luciferase activity was measured using a BioTek Cytation 3 equipped with Gen5 software (v3.08).

Protein Expression and Purification

The full-length cytosolic parasitic TgcPheRS and HscPheRS genes (α and β) were synthesized and codon optimized for optimal expression in E. coli cells. The genes were cloned together in a pETM11 vector respectively using Nco1 and Kpn1 restriction sites as described previously^1,2. This composite vector was transformed in the BL21 strain of E. coli; cultures were grown briefly at 37 °C until OD₆₀₀ reached 0.6–0.8, and protein expression was induced by adding 0.6 mM (IPTG) at 18 °C. After 18–20 h, cells were harvested via centrifugation at 5,000 g for 20 min, resuspended and lysed in binding buffer comprising 50 mM Tris–HCl (pH 8), 200 mM NaCl, 4 mM β-mercaptoethanol (βMe), 10% (v/v) glycerol, 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 0.1 mg/mL lysozyme by sonication. After centrifugation at 20,000 g for 45 min, the supernatant was loaded on a prepacked Ni-NTA column (GE Healthcare) and washed with 20 column volumes of binding buffer supplemented with 20 mM imidazole to eliminate impurities. The bound proteins were eluted using a concentration gradient of imidazole (0 to 1 M) using the AKTA-FPLC system (GE Healthcare). The eluted fractions containing protein were concentrated using 30-kDa cut-off centrifugal devices (Millipore) and purified by size exclusion chromatography using the HiLoad 60/600 Superdex 200 pg column (GE Healthcare) column in a buffer comprising 25 mM Tris (pH 8), 200 mM NaCl, 4 mM β-mercaptoethanol. Purity was confirmed via SDS PAGE gel, followed by pooling and storing of fractions at −80 °C for further use.

Thermal Shift Assays (TSA)

TSA experiments were conducted by diluting the purified TgcPheRS and HscPheRS enzyme in a buffer comprising 50 mM Tris (pH 7.5), 200 mM NaCl, 5 mM MgCl₂ and 2 X SYPRO orange dye (Life Technologies) as described earlier^{2, 3}. The purified enzyme (2 uM) was incubated with 20 μM of the test compounds at room temperature for 10min. The samples were heated from 25°C TO 99°C at a rate of 1°C min⁻¹ and the fluorescence signals of SYPRO^® orange dye were monitored using StepOnePlus ^™ quantitative real-time PCR system (Life Technologies). Mean values of derivative Tm from triplicates are presented, and analysis was done using Protein Thermal shift software (v1.3, Thermofisher).

Enzyme Assays

Enzyme inhibition assays were assessed using malachite green dye-based aminoacylation assays as previously described^{1, 2, 3, 4}. Reactions were carried out with 50nM of purified TgcPheRS and HscPheRS enzyme in the presence of 50 μM L-phenylalanine and 100 μM ATP, and 2 U/mL E. coli inorganic pyrophosphatase in a buffer containing 30 mM HEPES (pH 7.5), 50 mM MgCl_2, 150 mM NaCl, 30 mM KCl, 1 mM DTT. For Ic₅₀ determination, the test compounds were serially diluted (10-fold dilution) in 96-well clear, flat-bottom plates. The plates containing the reaction mixtures were incubated at 37°C for 2 h and terminated using malachite green dye. Absorbance at 620 nm were measured using SpectraMax M2 (Molecular Devices). Three independent replicates were performed and data sets were analyzed using graph pad Prism6.

Ex-vivo Bradyzoite differentiation assay.

CBA/CaJ mice (Strain 517#000654) from Jackson Laboratory were housed at Washington University School of Medicine in an approved facility. All animal studies adhered to ethical guidelines approved by the Institutional Animal Care and Use Committee. Production of chronically infected mice and isolation of tissue cysts was conducted using previously described protocols²⁴. In brief, CBA/CaJ mice were infected via oral gavage with 5−10 TgME49-EW strain²⁶ tissue cysts from the brain homogenate of previously infected mice. At 1−2 months post-infection, brains of mice infected with the strain were homogenized, and tissue cysts were isolated using Percoll gradients. To release bradyzoites, purified tissue cysts were treated with an acid-pepsin solution (170 mM NaCl and 60 mM HCl) and a freshly prepared pepsin solution (0.1 mg/mL in 1x PBS) for 10 min at 37°C. The reaction was stopped by adding a neutralization buffer (94 mM Na₂CO₃). Liberated bradyzoites were distributed into duplicate 6-well plates (technical replicates, one for constant compound treatment and other for 4 h treatment followed my compound free media named washout), each containing 5 mL of culture media. Each plate included a negative control (media with 0.1% DMSO) and pyrimethamine (2.0 μM). Compounds were tested at 3xEC₉₀ concentrations based on their in vitro tachyzoite growth inhibition (TgEC₅₀) assays (Table 2). After a 4 h treatment, compounds were removed from one set of plates by washing 3 times with PBS and replacing with compound-free D10 medium. Plates were then incubated at 37°C with 5% CO₂ for 12−14 days to form plaques. Plaques were fixed in 100% ethanol for 5 min, stained with 0.1% crystal violet for 10 min, rinsed with water, and air-dried. Plaque quantification was performed using a Nikon Eclipse TS2 microscope with a 4X objective, and the number of plaques was normalized to DMSO control as a percentage.

To test the effect of the compounds on intact cysts, replicate samples of Percoll-purified cysts (10 cysts/compound) were treated with 3xEC₉₀ concentrations in a 1 mL Eppendorf tube at 37°C for 4 or 24 h. Following treatment, cysts were washed three times with PBS, bradyzoites were released using an acid-pepsin solution, and the reaction was stopped with a neutralization buffer. Bradyzoites were then distributed into parallel 6-well plates in media with 3xEC₉₀ concentration compounds (Constant) or media without compounds (Washout). Plates were incubated at 37°C with 5% CO₂ for 12−14 days to form plaques. Plaques were quantified as described above.

In vivo efficacy studies.

Ethical statement

Female C57/BL6 mice (Strain #000654) were purchased from Jackson Laboratory and housed at Washington University School of Medicine in an AAALAC approved facility. All procedures followed ethical guidelines approved by the Institutional Animal Care and Use Committee at Washington University School of Medicine under Animal Welfare Assurance #D16–00245.

The compounds 12 and BRD7929 were formulated at 2 mg/mL in a freshly prepared compound resuspension buffer (20% PEG 400, 10% Vitamin E TPGS, 70% sodium acetate buffer (50 mM, pH 4.1), and 0.25 mg/mL sulfadiazine in deionized water (filtered sterilized). In the first experiment, five cohorts of 8 mice were infected with 300 TgME49Fluc tachyzoite parasites via intraperitoneal (IP) injection. Four days post-infection, one cohort, received 3 mg/kg 12 twice daily (BID), one cohort received 10 mg/kg 12 once daily (QD), one cohort received 10 mg/kg BRD7929 QD, one cohort received sulfadiazine (0.25 mg/mL in drinking water), and a control cohort received an equal volume of vehicle BID. In the 2^nd experiment, three cohorts of 8 mice were infected with 1000 TgME49Fluc tachyzoite parasites via IP injection. Four days post-infection, one cohort received 1 mg/kg 12 BID, one cohort received 3 mg/kg 12 BID and control cohort received an equal volume of vehicle BID. All mice were monitored for survival and weight loss every 2–3 days for 30 days post-infection. Blood samples (50–100 uL) were collected from surviving mice to test for seroconversion using a previously described ELISA assay based on whole tachyzoite lysate²⁴. Animals were sacrifices, and the brain was excised and homogenization in 1 mL of 1x PBS. Brain homogenates (2×100 μL/well) were seeded in 6-well plates containing confluent HFF in D10 media and monitored for 10–14 days for parasite growth and plaque formation. Plaques were fixed in 100% ethanol for 5 min, stained with 0.1% crystal violet for 10 min, rinsed with water, and air-dried. Presence of any plaque was performed using a Nikon Eclipse TS2 microscope with a 4X objective. Any parasite growth observed was further tested for resistance to the respective compound using Luciferase-based growth inhibition TgEC₅₀ assay defined earlier. In addition, immunocompromised interferon gamma receptor 1 knockout (B6.129S7-Ifngr1^tm1Agt/J) mice were bred locally at Washington University. Two experiments were conducted: the first experiment included 4 groups with 5 mice per group, and the second experiment included 3 groups with 6 mice per group. These studies were combined due to the sensitivity of this model and the rapid loss of mice, which would otherwise reduce the power of any statistical comparisons. Instead of waiting for 4 days post-infection, we initiated observations 1 day post-infection.

Serum Chemistry

Female C57/BL6 mice (Strain 517#000654) were purchased from Jackson Laboratory and housed at Washington University School of Medicine in an approved facility. All procedures followed ethical guidelines approved by the Institutional Animal Care and Use Committee. The 12 were prepared as define above. Three cohorts of 8 mice were used in this experiment, one cohort received 1 mg/kg 12 BID, one cohort received 3 mg/kg 12 BID and control cohort received an equal volume of vehicle BID for 7 days. Blood samples were collected at 24 h postdosing and analyzed by Washington University Division of Comparative Medicine Diagnostic Laboratory using the ACE Axcel^® Clinical Chemistry System.

Pharmacokinetics Studies

General methods for all (IV or PO) reported pharmacokinetic studies included here used three fasted animals per study group that were administered the test compound as a solution or suspension formulation. Male CD-1 mice were used. Blood and brain samples were collected at 1, 3, 8 hours if not specified. The blood samples were centrifuged to obtain the plasma; the plasma was stored at below −20 °C until analysis. Plasma concentrations were determined by liquid chromatography-tandem mass spectrometry (LC−MS/MS). The PK parameters were determined by noncompartmental methods using WinNonLin (v6.1 or higher version, Certara Inc.). For 4-day BID study of 12, the interval between doses within a day was 10 h.

Statistical Analysis

Statistical analyses were performed using Prism 10 (GraphPad Software, Inc.). Dose-response inhibition curves for parasite and host cell toxicity screens (EC₅₀ and CC₅₀ values) were generated using the “Log(inhibitor) vs. normalized response—Variable slope” function. Values are reported as means of three or more biological replicates.

Supplementary Material

Supplemental Figures and methods

Figure S1 SDS-PAGE analysis of purified PheRS enzymes.

Figure S2 Time dependent inhibition of T. gondii bradyzoite growth within intact cysts.

Figure S3 PK profile of top leads.

Figure S4 Serology testing by ELISA.

Table S1 Comparison of EC₅₀, IC₅₀ and ΔTm for PheRS enzymes.

Table S2 Pharmacokinetics Studies of top lead compounds

Supplementary Experimental section

Compound synthesis schemes

NMR spectra of final compounds

HPLC-MS traces of lead compound 12

Molecular Strings Formulae

Abbreviations used:

DMSO

dimethylsulfoxide

DHFR

dihydrofolate reductase

EDTA

ethylenediaminetetraacetic acid

HFF

human foreskin fibroblasts

HepG2

human hepatocellular carcinoma cell

THP-1

human monocytic tumor cell

nLuc

nano luciferase

Fluc

firefly luciferase

PK

pharmacokinetics

TSA

Thermal Shift Assays

SAR

structure activity relationship

PheRS

phenylalanine tRNA synthetase

L-Phe

L-phenylalanine