Discovery of New Anti-cryptosporidial Structures from Natural Products and Potential Synergistic Anti-cryptosporidial Activity by Drug Combinations

Cryptosporidium parvum is a zoonotic parasitic protist and the causative agent of cryptosporidiosis in humans and animals. The main symptom of cryptosporidiosis is diarrhea, which can be severe and life-threatening in immunocompromised patients and young animals. However, current therapeutics are limited. In humans, nitazoxanide (NTZ) is the only FDAapproved treatment for immunocompetent patients. In animals, only halofuginone lactate (Halocur) is approved in a few countries for veterinary use in calves and lambs. Additionally, both NTZ and Halocur are not 100% effective against cryptosporidiosis. Therefore, there is an urgent demand for developing more effective chemotherapies. The ultimate goal of this study is to discover potential new anti-cryptosporidial therapeutic options. Towards achieving this goal, we conducted phenotypic screening to identify new anti-cryptosporidial structures from 800 natural products and to explore combination of drugs with different targets for potential anti-cryptosporidial synergistic effect. Both approaches employed an in vitro model of infection by C. parvum. For natural products screening, we have identified 16 top hits with anti-cryptosporidia efficacies (ECv50 values from 0.122 to 3.940 μM) and cytotoxicity to host cell (TCv50 values from 6.31 to >100 μM), showing low to submicromolar anti-parasitic activity in vitro. Among them, three compounds with sub-micromolar ECv50 values (i.e., cedrelone, deoxysappanone B 7,4'-dimethyl ether [Deox B 7,4] and baicalein) were further investigated for their effectiveness on various parasite developmental stages in vitro. Cedrelone [CAS # 1254-85-9] and baicalein [CAS # 491-67-8] were more effective than Dexo B 7,4 [674786-37-9] when treating parasite for shorter periods of time, but all three compounds could kill the parasite irreversibly. For drug combination study of 9 pairs using 6 drugs, preliminary data indicated that the “paromomycin + vatalanib”, “paromomycin + vorinostat” and “paromomycin + triacsin C” produced synergistic effect. These observations pointed out a new direction to potentially synergize the anti-cryptosporidial efficacy and/or reduce the adverse effects of drugs at therapeutic doses. In summary, we have discovered a set of new structures from natural products with excellent anti-cryptosporidial efficacies in vitro for future drug design and development, and identified new options in combination therapy against cryptosporidiosis in humans and animals