Mechanistic insights into the activity of Benzoxaboroles against <i>Cryptosporidium parvum</i> : CPSF3 targeting and resistance implications

Abstract Benzoxaboroles are emerging as promising treatments against a broad range of protozoan parasites, including Trypanosoma , Leishmania , Plasmodium , Toxoplasma , and Cryptosporidium . We previously demonstrated that the benzoxaborole compound AN3661 inhibits the endonuclease activity of CPSF3 in Cryptosporidium hominis , likely by disrupting pre-mRNA processing, which in turn limits parasite growth. In this study, we further explored its mode of action and found that a specific mutation (Y385N) in CPSF3 of C. parvum confers strong resistance to AN3661. Interestingly, this mutation does not affect the parasite’s sensitivity to two other benzoxaboroles, AN13762 and AN7973, which are also thought to target Cp CPSF3. All three compounds interfered with mRNA processing in C. parvum , consistent with inhibition of CPSF3 complex activity, and showed parasiticidal activity, especially during the late stages of merogony, blocking parasite egress. Two of them also impaired gamogony. In T. gondii , AN7973 remained effective against several strains carrying mutations within TgCPSF3 that confer resistance to AN3661 and AN13762, suggesting that it might represent an alternative chemotype targeting CPSF3 with the potential to overcome resistance. Notably, AN7973 successfully controlled severe infection in susceptible mice challenged with the AN3661-resistant C. parvum strain carrying the CpCPSF3 Y385N mutation. Finally, we extended the known antiparasitic spectrum of AN3661 and AN7973 to include Eimeria tenella and Giardia duodenalis , two important pathogens in veterinary and human health. Altogether, our findings refine the understanding of CPSF3-targeting benzoxaboroles, identify alternative chemotypes with the potential to bypass resistance, and support their potential use in combination therapies to delay or prevent the emergence of drug resistance. Author summary Protozoa are single-celled parasites that cause significant morbidity and mortality worldwide, affecting both humans and animals. The development of new targeted therapies with highly selective compounds requires a detailed understanding of their mode of action and precise interactions with parasite targets. Benzoxaboroles are a potent class of molecules active against Cryptosporidium , with compound AN3661 known to inhibit the endonuclease activity of the Cleavage and Polyadenylation Specificity Factor 3 (CPSF3) in Cryptosporidium hominis . Here, we show that this inhibitory effect is critical during both asexual and sexual developmental stages and that tyrosine at position 385 of C. parvum CPSF3 plays a key role in its inhibition. A mutation at this site confers resistance to AN3661 both in vitro and in vivo , but not to two other benzoxaboroles, AN13762 and AN7973. Notably, all three compounds disrupted pre-mRNA processing in Cryptosporidium , consistent with inhibition of the CPSF3 complex. Using Toxoplasma , a related protozoan that allows more efficient genetic manipulation, we found that none of the six mutations conferring resistance to compounds AN3661 and AN13762 conferred resistance to compound AN7973. AN7973, which strongly inhibits both C. parvum and T. gondii , may therefore represent an alternative chemotype targeting CPSF3. Furthermore, we demonstrated that the antiparasitic spectrum of AN3661and AN7973 extends to include E. tenella and G. duodenalis , two important pathogens in veterinary and human health. Altogether, our results refine the understanding of three CPSF3-targeting benzoxaboroles in Cryptosporidium and identify compound AN7973 as capable of overcoming resistance to AN3661, thereby supporting the rationale for combination therapies to prevent the emergence of drug resistance.

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