Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites

Abstract

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Keywords: Plasmodium falciparum; RPS13Δ; Toxoplasma gondii; cytochrome bc1; nanoformulation; structure-guided design; tetrahydroquinolone; transcriptomics.

Figure 1

Characteristics and effects of compounds on inhibition of Toxoplasma gondii replication and enzyme activity, and Structure Activity Relationship analysis. (A) Cytochrome b/c inhibitor code, Chem Draw structure, solubility in PBS 7.4, toxicity against HFF, predicted half-life, and inhibitory effect of compounds on RH strain tachyzoites and EGS strain bradyzoites in vitro and saffarine O assay enzyme activity. PBS Sol/Toxicity pH7.4 refers to solubility of the compound in Phosphate Bufferred Saline (PBS) at pH 7.4. Toxicity refers to the highest concentration tested that does not show toxicity to Human Foreskin Fibroblast (HFF) in tissue culture in WST assay; T1/2 (H) refers to the predicted half-life in human liver microsomes; T1/2 (M) refers to the predicted half-life in mouse liver microsomes. Tachy/Brady IC50 was determined in studies in which cultures of parasites in HFF were treated with varying concentrations of the compound and there was 50% inhibition of the replication (number) of parasites. Parasites were RH-YFP expressing tachyzoites (Tachy) and EGS (Brady) strains. Studies of effects of inhibitors on HFF or on T. gondii tachyzoites were performed with triplicate wells in at least 2 biological replicate experiments. Studies of effects on bradyzoites were performed at least twice in at least 2 biological replicate experiments. Compounds with much less inhibition of mammalian than T. gondii cytochrome bc, relative to JAG21 effect on parasite enzyme, in the saffarine enzyme assay (indicated by **) provide potential to further develop compounds, if unanticipated toxicity occurs from JAG21. (B) Structure Activity Relationship analysis (SAR). The effects of changing R1 as 7-Et, 7-Me, 6-CF₃, or 6-Me on activity against T. gondii RH strain tachyzoites, solubility, and stability were compared in the SAR. Color Key in (B) Activity: Green <50 nM, Red > 1 μM; Solubility in 100 mM Phosphate Buffer (pH 7.4): Amber>10 μM, Red <10 μM; Metabolic Stability: Green >120 min, Amber 60–120 min, red <60 min. SAR panel displays only representative structures and trends within the JAG compound series. JAG21 (blue font) is highly active, has the longest predicted half-life for humans of initial compounds tested (green), combined with improved solubility, no hERG liability, and predicted capacity to cross the blood brain barrier (BBB). Definitions of ADMET terminology are in the Materials and Methods. In summary, in the SAR overall, nitrogen atoms were not tolerated in aryl ring marked by green c, and the 4-position was optimal for phenol substituent. Compound name with “0” or no “0” between letters and number, e.g., JAG21 or JAG021, refer to the same compound. This is throughout the manuscript.

Scheme 1

Synthesis of hit compound 7, also known as MJM170 (McPhillie et al., 2016). Synthetic scheme inspired by the route to endochin-like quinolones (ELQs) reported by Doggett et al. (2012).

Scheme 2

Synthetic route to analogs of 7 (MJM170) via route A or route B. Route A is the original route to analogs but is linear and involves a tricky Suzuki step to intermediate 12 from intermediates 4 and 10. Route B allows quicker access to analogs since intermediate 15 can be made in larger quantities and derivatives can be synthesized via the Chan-Lam reaction to give final intermediate 12 by varying the boronic acid 16.

Figure 2

JAG21 is potent in vitro against Toxoplasma gondii, tachyzoites and bradyzoites, and multiple drug resistant strains of P. falciparum. (A) JAG21 is effective against RH-YFP tachyzoites, and does not harm human cells. Potent effect of JAG50 is also shown. A representative experiment is shown. N = triplicate wells in at least 2 biological replicate experiments. Relative fluorescence units are shown on the vertical axis, where decrease in fluorescence compared to diluent DMSO in media control indicates parasite inhibition (*p < 0.05). Horizontal axis indicates different treatment conditions: This shows results of testing of fibroblasts in media (HFF), DMS0 control, positive control pyrimethamine and sulfadiazine(P/S), and concentrations of JAG21 and JAG50 utilized. Differences were not statistically significant in the cytotoxicity assay (data not shown). (B) JAG21 is effective against EGS bradyzoites. Effect of JAG21 in reducing bradyzoites in HFF by parasite strain EGS. HFF were infected by EGS and treated with JAG21 at concentrations indicated. Slides were stained with Dolichos Biflorus Agglutinin conjugated with FITC (which stains the cyst wall) and DAPI, and observed with fluorescence microscopy. The red arrows point to the Dolichos enclosed organisms formed in tissue culture. These were eliminated with treatment with JAG21. This experiment was performed >4 times. These experiments were performed with 3 different observers reviewing slides at the microscope quantitating fields for each condition. Slides were also scanned and the scans of the slides were reviewed so all fields in the entire slide were noted to be consistent. (C) Synergy of JAG21 and atovaquone against Rh-YFP tachyzoites in vitro. Isobologram comparing JAG21, atovoquone, and JAG21 plus atovaquone demonstrates synergy. (D) THQs effective against drug resistant P. falciparum. Dose-response phenotypes of a panel of P. falciparum parasite lines. IC50 values were calculated using whole-cell SYBR Green assay and listed as mean ± standard deviation of three biological replicates, each with triplicate measurements. The D6 strain is a drug sensitive strain from Sierra Leone, the TM91-C235 strain is a multi-drug resistant strain from Thailand, the W2 strain is a chloroquine resistant strain from Thailand, and the C2B strain is a multi-drug resistant strain with resistance against atovaquone. (E) Solubility and Stability in human and mouse liver microsomes comparing MJM 170, JAG21, and JAG50. Performed by Chem Partners. (F) JAG21 CYP450 Inhibition, CACO-2, hERG, PPB, BBB (MDCK-MDK1) efflux analyses. These were performed by Chem Partners and are as defined in the section Materials and Methods. RG38 is a structurally related inactive THQ analog.

Figure 3

Effect of JAG21, and other THQ compounds on mitochondrial functions of Toxoplasma gondii, Plasmodium falciparum and HFF-hTE RT (A). Maximum mitochondrial membrane depolarization of JAG21, JAG39, JAG46, JAG47, JAG50, and Atovaquone (4 μM) and FCCP (5 μM). Digitonin was added where indicated by the arrow to permeabilize cells and permit a necessary mitochondrial substrate (Succinate) to reach intracellular organelles. The addition of the indicated compounds is shown by the second arrow. (B) Quantification of the depolarization shown in (A). The relative depolarization of each compound was normalized to the depolarization by FCCP which was considered 100% depolarization. (C) Effect of various concentrations of JAG21 and Atovaquone on the mitochondrial membrane potential measured as in (A). The first arrow indicates digitonin addition and the second arrow indicates the addition of compounds at the specified concentration. (D) Quantification of the depolarization measured in (C). The relative depolarization of each compound was normalized to the depolarization by FCCP (100%). (E) Mitochondrial membrane depolarization of HFF-hTERT in suspension by JAG21 and atovaquone. The first arrow indicates the addition of digitonin, and the second arrow indicates addition of the indicated compounds at the indicated concentration. (F) Quantification of the depolarization measured in (E). The relative depolarization of each compound was normalized with the depolarization by FCCP, which was considered 100%. (B,D,E) X ± S.D., N = 3 independent experiments. Statistical analysis (unpaired student t-test) was performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA). **P < 0.01. ***P < 0.001. (G) JAG21, JAG99, and MJM210 (1 μM) inhibited P. falciparum cytochrome c reduction. Vehicle (DMSO)/atovaquone (1 μM) were negative/positive controls, 1,290 respectively. X ± S.D., N = 4 independent experiments.

Figure 4

Binding studies of JAG21 to bovine bc1. (A) Bovine Cytbc1 activity assays showing 36 and 63% inhibition at 0.1 and 1 μM concentration of JAG21, respectively. N = at least 2 biological replicate experiments with similar results. (B) The Cytbc1 structure presented in cartoon style with clear omit (Fo-Fc) electron density map for the bound JAG21 compound only in the Q_i site showing selectivity within the binding pocket. Qi and Qo sites are marked by black ellipsoids. (C) The bound JAG21 compound (orange) within the Qi site with corresponding (2Fo-Fc) electron density map contoured at 1 σ level as gray mesh. The residues which make close interactions with the bound inhibitor are shown in stick format and labeled. (D) 2D pharmacophore analysis of JAG21 binding pocket produced using Ligplot+ LS-2011. Hydrophobic interactions are shown as red spikes, hydrogen bond with Ser35 is shown by green dashes. (E) Cryo-EM derived structure of the Cytbc1 bound JAG021 structure with corresponding density map contoured at 3 σ level suggesting two different positions for the head group represented by two regions of density shown as yellow mesh. The Cytbc1 structure bound to the pyridone GSK932121 (PDB:4D6U) (F) and quinolone MJM170 (PDB:5NMI) (G) in the Q_i site. Haem and compounds are shown as colored sticks, Fe ion as orange sphere and hydrogen bonding as black lines. Hydrogen bonding with Ser35 is shown as black dashes. Terms JAG021 and JAG21 used interchangeably for this same compound.

Figure 5

JAG21 is a mature lead that protects against Toxoplasma gondii and Plasmodium berghei in vivo. (A) JAG21 treatment for 14 days protects against T. gondii tachyzoites in vivo. Tachyzoite challenge with Prugneaud luciferase parasites imaged with leuciferin using IVIS demonstrates that treatment with JAG21 eliminates leuciferase expressing parasites and leads to 100% survival of JAG21 treated infected mice. No cysts were found in brains of mice at 30 days after infection when they have been treated with JAG21 for the first 14 days after infection. There were 2 biological replicate experiments with 5 mice per group with similar results. (B) JAG21 and JAG21 plus tafenoquine markedly reduce Me49 strain brain cyst numbers in vivo in Balb/C mice at 30 days after infection. Parasites were quantitated by scanning the entire immunoperoxidase stained slide in an automated manner and by two observers blinded to the experimental treatment using microscopic evaluation. In each of two experiments, the numbers of mice per group were as follows: Experiment 1 had 4 diluent controls, 5 JAG21, 4 JAG21/Tafenoquine treated mice; and Experiment 2 had 5 diluent controls, 5 JAG21, 3 JAG21/Tafenoquine treated mice. Immunoperoxidase staining was performed. Parasite burden was quantitated using a positive pixel count algorithm of Aperio ImageScope software. Positive pixels were normalized to tissue area (mm²). Quantification was by counting positive pixels per square area. The entire brain in one section was scanned for each mouse. The parasite burden was quantitated as units of positive pixels per mm². The average ± S.E.M. numbers of mm² per slide quantitated was 30.2±1.6 mm² per mouse for this quantification. Each high power field of view shown in C is ~0.02 mm² per field of view. A representative single experiment is presented and the data from the two experiments analyzed together also demonstrated significant differences between the untreated and treated groups (p < 0.01; Supplemental Figure 1). (C) Microscopic evaluation of the slides reveal effect of JAG21 and JAG21 plus tafenoquine having the same pattern as the automated quantitation of immunoperoxidase stained material. There are usual appearing cysts in the DMSO control untreated mice as shown in the top panels, and rare cysts in the treated mice with most of the brown material appearing amorphous (bottom panels). (D) JAG21 nanoformulation dosages administered to P. berghei infected C57Bl6/albino mice compared with vehicle control. Design of single dose and 3 day dose experiments. (E) JAG21 nanoformulation cures P. berghei sporozoites (left panel), blood (middle panel), and liver stages, leading to 100% survival (right panel). This is with oral administration of a single dose of 2.5 mg/kg or 3 doses at 0.625 mg/kg. Single dose causal prophylaxis in 5 C57BL/6 albino mice at 2.5 mpk dosed on day 0, 1 h after intravenous administration of 10,000 P. berghei sporozoites. Shown is 3 dose causal prophylaxis treatment in 5 C57BL/6 albino mice at 0.625 mpk dosed on days −1, 0, and +1. Representative figure showing survival (right panel), luminescence (left panel), and parasitemia quantitated by flow cytometry (middle panel) for 5 mg/kg.

Figure 6

Toxoplasma gondii ΔRPS13 transcriptome during Primary Human Brain Neuronal Stem Cell (NSC) infection and in-vivo susceptibility to JAG21 and TAF treatment are reminiscent of literature findings with malaria hypnozoites. (A) P. cynomolgi-T. gondii best reciprocal match genes significantly upregulated (red) or downregulated (blue) in P. cynomolgi hypnozoites compared to liver-schizont stage and in ΔRPS13 after downregulation of rps13 gene expression (p ≤ 0.05, FDR ≤ 0.2). (B) Gene-set enrichment analysis of ΔRPS13 +/– Tc. Blue and red nodes denote gene-sets enriched in presence or absence of Tc, respectively. Node diameters are proportional to number of genes belonging to corresponding gene-sets. Edge thickness is proportional to number of genes shared between connected nodes. (C) Survival rate of mice infected with 100,000 ΔRPS13 followed by treatment with diluent, JAG21, tafenoquine (TAF) or the two together (JAG21/TAF). Then tetracycline was added at 14 days. The combination of the two compounds resulted in improved time of survival (p < 0.03, Experiment 1; p = 0.08 Experiment 2, p = 0.002 Experiment 1+2). The full data are presented in the box below the image in (C). (C) Rx refers to treatment of mice with diluent (DMSO), Tafenoquine (TAF), or JAG21, or JAG21 and TAF. ΔRPS13 is referred to as RhRPS13Δ in the title of the box in (C). In Supplemental Figure 2, histological preparations that are immunoperoxidase stained for T. gondii antigens from a pilot study were prepared (Supplemental Figure 2). These are images, in Supplemental Figure 2 of liver and spleen from IFN γ receptor knock out mice without treatment on days 7 and 14 after infection. In those mice without any treatment there was amorphous brown immunoperoxidase stained material in Supplemental Figure 2A. When tetracycline (aTet) was administered on day 14 after infection in drinking water, with tissues obtained and immunostained for T. gondii antigens from mice that died or became very ill, organisms that were clearly recognizable could be seen (Supplemental Figures 2B–E). Design of the treatment experiment with control DMSO diluent, JAG21 alone, Tafenoquine alone (TAF) or the two together (JAG/TAF) with full data for each of the groups and with the composite analysis from replicate experiments, including numbers of mice, are shown in Supplemental Figures 2C,D. Supplemental Figures 2C,D shows prolongation of survival time, but there is not durable protection against ΔRPS13 in these immune compromised mice treated with JAG21/TAF as described. This is summarized in C to demonstrate early prolongation of survival time with the detailed data in Supplemental Figure 2. (D) Gene ontology enrichment analysis of ΔRPS13 +/–Tc. Node and edge conventions are the same as in (B). There were at least 2 biologic replicates of each experiment.

Figure 7

Oral nanoformulation of JAG21 potently protects against 2000 highly virulent RH strain tachyzoites given intraperitoneally. (A) Following sonication produces nanoparticles of ~2.86 μM. (B) Single oral dose of 10 mg/kg reduced intraperitoneal tachyzoites measured by RH YFP expression and counting with hematocytometer (p < 0.03). (C) Three daily 10 mg/kg doses markedly and significantly reduces intraperitoneal parasite burden measured as fluorescence and by hematocytometer on the fifth day (p < 0.001). No compound was administered after the third day. N = at least 2 biological replicate experiments with 5 mice per group with similar results.