Cryptosporidium lysyl-tRNA synthetase inhibitors define the interplay between solubility and permeability required to achieve efficacy

Abstract

Cryptosporidiosis is a diarrheal disease caused by infection with Cryptosporidium spp. parasites and is a leading cause of death in malnourished children worldwide. The only approved treatment, nitazoxanide, has limited efficacy in this at-risk patient population. Additional safe therapeutics are urgently required to tackle this unmet medical need. However, the development of anti-cryptosporidial drugs is hindered by a lack of understanding of the optimal compound properties required to treat this gastrointestinal infection. To address this knowledge gap, a diverse set of potent lysyl-tRNA synthetase inhibitors was profiled to identify optimal physicochemical and pharmacokinetic properties required for efficacy in a chronic mouse model of infection. The results from this comprehensive study illustrated the importance of balancing solubility and permeability to achieve efficacy in vivo. Our results establish in vitro criteria for solubility and permeability that are predictive of compound efficacy in vivo to guide the optimization of anti-cryptosporidial drugs. Two compounds from chemically distinct series (DDD489 and DDD508) were identified as demonstrating superior efficacy and prioritized for further evaluation. Both compounds achieved marked parasite reduction in immunocompromised mouse models and a disease-relevant calf model of infection. On the basis of these promising data, these compounds have been selected for progression to preclinical safety studies, expanding the portfolio of potential treatments for this neglected infectious disease.

Figure 1. A balance between solubility and permeability maximizes efficacy in a NOD SCID Gamma mouse model of cryptosporidiosis.

(A) Schematic of Biopharmaceutical Classification System (BCS). (B) Aqueous solubility and Parallel Artificial Membrane Permeability Assay (PAMPA) measurements of tool compounds from each BCS class. Data are means of two technical replicates. Solubility and permeability cut-offs were defined at 100 μM and 30 nm/s respectively. Raw data reported for all tool compounds in Table S1. (C) NOD SCID Gamma mice were infected with wild type C. parvum. Vehicle, positive control, or tool compound was administered via oral gavage starting day 7 post infection (PI) for 7 days (gray box indicates treatment period). Fecal samples were collected on days indicated by arrows. Fecal samples were collected from individual mice (4 animals per treatment group) and analyzed by qPCR to quantify parasite shedding (oocysts/g). (D) Log reduction in oocyst shedding measured on day 14 PI vs day 21 PI for tool compounds (colored by BCS class) dosed orally at either 30 or 50 mg/kg twice a day (BID) for 7 days in NOD SCID Gamma mice. Most compounds dosed at 50 mg/kg; DDD212, DDD352, DDD478, DDD583, DDD582 dosed at 30 mg/kg. Mean oocyst shedding was determined for each group of n=4 mice, for all compounds tested. Log reduction in oocyst shedding was calculated as log (mean oocysts on day 7) – log (mean oocysts on day 14 or 21). Two tool compounds that sustain reduction in parasite shedding for a week beyond treatment are labeled in bold. Independent experimental repeats with additional doses of tool compounds reported in Fig. S4.

Figure 2. Minimum efficacious dose of DDD489 and DDD508 is 30 mg/kg in NOD SCID Gamma mice.

Late lead compounds were administered to NOD SCID Gamma mice as described in Fig. 1C. (A and B) Mice were administered vehicle (white), paromomycin at 2000 mg/kg QD (positive control; black), and DDD489 (A, gray) or DDD508 (B, gray) at 30 mg/kg BID. Representative experiment shown. Independent experimental repeats with additional doses reported in Fig. S5. Bar height indicates average for each treatment group (n=4 mice per cage for all compounds tested), points indicate individual animals (average of three technical replicates plotted). Y-axes begin at limit of quantitation (LOQ).

Figure 3. Low solubility or permeability results in low oral bioavailability in mice for late lead compounds.

Mean free blood concentration time profiles after a single oral dose (10 mg/kg) of DDD489 (A) or DDD508 (B) to female BALB/c mice (n=3 mice per group for all compounds tested). Data are mean ± SD. Total drug concentrations were corrected for fraction unbound (fu) (DDD489 fu: 0.16; DDD508 fu: 0.6) assuming blood-to-plasma ratio of 1. Dotted lines indicate C. parvum EC₉₀, calculated from average EC₅₀ and hill slope (DDD489: EC₅₀ 0.043 μM, hill slope 2.7; DDD508: EC₅₀ 0.13 μM, hill slope 2.1). Both compounds have low oral bioavailability and low clearance (DDD489: F=7%, Clb=17% LBF; DDD508: F=6%, Clb=27% LBF).

Figure 4. Late leads DDD489 and DDD508 are effective in key cryptosporidiosis mouse models.

Efficacy of late lead compounds was assessed in an Interferon-Gamma KO model (acute model of cryptosporidiosis) and in chronically infected NOD SCID Gamma mice. Fecal samples were collected and pooled from each cage of mice; parasite shedding was quantified via NanoLuciferase assay (relative luminesce units, RLUs/g; average of three technical replicates plotted). Y-axis begins at LOQ. (A) Interferon-Gamma KO mice were infected with transgenic C. parvum that express NanoLuciferase. Treatments were administered by oral gavage starting on day 6 PI for 7 days (gray box indicates treatment period; n=5 mice per cage for all compounds tested). (B) Mice treated with vehicle (white), DDD706 (positive control, black), or DDD508 (teal). Associated PK data and infection quantification of individual mice reported in Fig. S6 and S7, respectively. ^†Mice were culled due to weight loss. (C) NOD SCID Gamma mice were infected with transgenic C. parvum that express NanoLuciferase. Fecal samples collected, pooled, analyzed, and graphed as in B (n=4 mice per cage for all compounds tested). Mice were treated when infection was chronic and high (RLUs/g >10⁹ for over a week). Treatment was administered via oral gavage starting on day 36 PI for 7 days (gray box indicates treatment period). (D) Mice treated with vehicle (white), control DDD706 (black), or DDD489 at 50 mg/kg BID (purple).

Figure 5. DDD489 and DDD508 reduce parasite shedding and diarrheal scores in calves.

(A) Neonatal calves are a large animal model and natural host model of cryptosporidiosis. Calves were enrolled in the study at the time of birth and randomly assigned to treatment or control groups (n=7 calves per group). Calves were challenged with wild type oocysts at two days old (0 days PI). Treatment with vehicle or late lead compound was administered orally at 15 mg/kg BID, starting at day 1 PI for 7 days (gray box indicates treatment period). Fecal samples were collected from individual calves daily starting the day prior to infection and ending day 21 PI. (B and C) Calves were treated with vehicle (white), DDD489 (B, black) or DDD508 (C, black). Associated PK data shown in Fig. S10. Parasite shedding was quantified by qPCR and by microscopy; mean values from each group plotted (see Fig. S8 and S9 for microscopy and for individual animal data for qPCR and microscopy). Y-axes begin at LOQ. (D and E) Mean fecal consistency scores recorded during treatment period (day 1 to day 7 PI) for vehicle and treated animals. Diarrhea was scored based on fecal consistency (for scoring guide see Table S4). Points represent individual animals, with mean and 95% confidence intervals plotted as horizontal gray lines. P value was determined using the unpaired, single-sided student’s t test (D) p=0.0375 (*) (E) p=0.002 (**)

Figure 6. DDD489 and DDD508 bind in a similar pose in the ATP site of CpKRS.

(A and B) Crystal structures of DDD489 (A, blue, PDB ID 8R2A) and DDD508 (B, green, PDB ID 8S00) bound to the ATP site of CpKRS. Hydrogen atoms shown in calculated positions. Dotted lines represent hydrogen bonding (yellow), pi stacking (blue), and pi-cation (green) interactions. Key residues labeled for clarity. (C) Overlay of DDD489 (blue) and DDD508 in the ATP site of CpKRS. (D) Docked pose of DDD489 (blue) in the mutated KRS-A309L strain showing the anticipated clash between the mutated Leu309 residue (orange) and the trifluoromethyl-substituted cyclohexyl ring when bound in the hydrophobic ribose pocket.