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

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 ( h PDE-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 h PDE-V inhibitors sildenafil and the 4-aminoquinoline compound 7a do not affect Cryptosporidium . C. parvum expresses one PDE ( Cp PDE1) continuously during asexual growth, the inhibited life stage. According to homology modeling and docking, the lead compounds interact with Cp PDE1. Bulkier amino acids (Val900 and His884) in the Cp PDE1 active site replace alanines in h PDE-V and block sildenafil binding. Supporting this, sildenafil kills a CRISPR-engineered Cryptosporidium Cp PDE1 V900A mutant. The Cp PDE1 mutation also alters parasite susceptibility to pyrazolopyrimidines. Cp PDE1 is therefore a validated pyrazolopyrimidine molecular target to exploit for target-based optimization for improved anticryptosporidial development. Using phenotypic screening followed by optimization of side activities, the authors here identify pyrazolopyrimidine phosphodiesterase (PDE) inhibitors as anti-cryptosporidial drug leads. Humanizing a Cryptosporidum PDE by CRISPR indicates they target the parasite enzyme.