Identification of potent and orally efficacious phosphodiesterase inhibitors in Cryptosporidium parvum-infected immunocompromised male mice

Abstract

Cryptosporidium parvum and C. hominis are parasites that cause life-threatening diarrhea in children and immunocompromised people. There is only one approved treatment that is modestly effective for children and ineffective for AIDS patients. Here, screening 278 compounds from the Merck KGaA, Darmstadt, Germany collection and accelerated follow-up enabled by prior investigation of the compounds identifies a series of pyrazolopyrimidine human phosphodiesterase (PDE)-V (hPDE-V) inhibitors with potent anticryptosporidial activity and efficacy following oral administration in C. parvum-infected male mice. The lead compounds affect parasite host cell egress, inhibit both C. parvum and C. hominis, work rapidly, and have minimal off-target effects in a safety screening panel. Interestingly, the hPDE-V inhibitors sildenafil and the 4-aminoquinoline compound 7a do not affect Cryptosporidium. C. parvum expresses one PDE (CpPDE1) continuously during asexual growth, the inhibited life stage. According to homology modeling and docking, the lead compounds interact with CpPDE1. Bulkier amino acids (Val900 and His884) in the CpPDE1 active site replace alanines in hPDE-V and block sildenafil binding. Supporting this, sildenafil kills a CRISPR-engineered Cryptosporidium CpPDE1 V900A mutant. The CpPDE1 mutation also alters parasite susceptibility to pyrazolopyrimidines. CpPDE1 is therefore a validated pyrazolopyrimidine molecular target to exploit for target-based optimization for improved anticryptosporidial development.

Fig. 1. Screening results and prioritization considerations.

The shown phosphatidyl-inositol-3-kinase inhibitor (PI3Ki), phosphodiesterase-5 inhibitor (PDEi) and IkappaB kinase 2 inhibitor (IKK2i) compounds were prioritized for follow-up based on confirmatory anticryptosporidial EC₅₀ data, existing safety and pharmacologic data, and the availability of chemical analogs for initial follow-up studies.

Fig. 2. PDEi efficacy in established murine C. parvum infection.

The indicated compounds were tested for efficacy in established cryptosporidiosis in male NSG mice. C. parvum infection was allowed to progress for either 7 days (A) or 14 days (B) prior to oral administration of 50 mpk twice daily of the indicated compounds or 1000 mpk twice daily of paromomycin (positive control) by oral gavage for 7 days. Parasite shedding in the feces was determined at the onset of treatment (D1), completion of treatment (D8), and one week after completion of treatment (D15). Data are the mean and SEM (n = 4 mice per experimental group) of parasite fecal shedding per mg of feces, and data points below the limit of qPCR detection are shown on the x-axis. p values vs. vehicle control determined by Kruskal–Wallis test are shown. Source data for (A) and (B) are provided as a Source Data file.

Fig. 3. Mouse PK data for compound PDEi2.

A Time courses of total drug concentration determined in plasma with single dose IV and oral administration at indicated doses. B Time courses of total drug concentration determined in washed intestinal tissue segments with single dose 10 mg/kg oral administration. Data for (A) and (B) are mean and SEM (n = 3 mice per experimental group where SEM shown; n = 2 mice per group where no SEM shown). Source data for (A) and (B) are provided as a Source Data file.

Fig. 4. In vitro rate of parasite elimination.

Rate-of-action curves for (A) nitazoxanide (NTZ) and (B) PDEi5 showing elimination of C. parvum from infected HCT-8 cells. One-phase exponential decay curves were fit for NTZ and PDEi5. Data are the means and SD for 4 culture wells per condition, normalized for each time point to the mean of 28 vehicle (DMSO) control culture wells. Results from one of two experiments shown. Source data are provided as a Source Data file. A No exponential decay curve could be fit for NTZ at the EC₉₀ which was the highest non-toxic concentration. Blue (DMSO); black (NTZ EC₉₀). B Rapid parasite elimination by PDEi5. Curves are shown for 3×EC₉₀ (gray) and 6×EC₉₀ (cyan) (the highest concentration tested), or the DMSO control (blue).

Fig. 5. Overlay of lowest energy pose from induced fit docking of PDEi5 in the CpPDE1 model (gold) with hPDE-V (purple; pdb 1UDT).

Only the seven of the 18 total residues within 4 Å of the PDEi5 ligand that differ between the two proteins are shown, along with Val900 (5 Å). The ligand binding pocket of CpPDE1 is shown as a gray surface. A Induced fit docked pose of PDEi5. B Overlay of sildenafil from hPDE-V structure (pdb 1UDT). Arrows indicate steric clashing with Val900 and His884 preventing sildenafil from obtaining the same docking pose in CpPDE1 vs. hPDE-V.

Fig. 6. Genetic validation of CpPDE1 as a drug target.

A CRISPR strategy for humanizing CpPDE1. A Val900Ala mutation and nano-luciferase/Neo resistance cassette were incorporated in the CpPDE1 locus. Transgenic control parasites were also made. B PCR (primers P1 and P2 in (A)) confirming modification of CpPDE1 (representative of 2 experiments). C Sanger sequencing confirming the designed mutation. For (D–F), dose response assays were conducted with the indicated inhibitor and CpPDE1 Val900Ala, CpPDE1 transgenic wt, and C. parvum(Iowa). Graphs show the means of 4 culture wells per condition (one of two experiments per inhibitor). Orange = CpPDE1 Val900Ala; gray = transgenic wt; black = C. parvum(Iowa). D Sensitization of the CpPDE1 Val900Ala parasite to sildenafil. E Effect of Val900Ala mutation on growth inhibition by PDEi5. F Effect of Val900Ala mutation on growth inhibition by PDEi2. Source data for all graphs are provided as a Source Data file.