Cryptosporidium is a parasite that affects both the very poor and the very rich. It can cause severe diarrheal infections in malnourished children and in people whose immune systems are compromised, for example, by HIV.
But since the end of November alone, it has also been linked to outbreaks of foodborne illnesses in such bucolic places as the Alpes-Maritimes, a region of France that includes part of the Cote d’Azur, and Sweden, whose public health care system ranks third only to that of its neighbors Norway and Finland in a 2019 Pew study.
Recent research that uncovered the mechanism of action of a boron-based oxaborole in controlling Cryptosporidium infection could further spur development of those compounds for treating infections by other parasite species and provides a target for de novo discovery of antiparasitic drugs and treatments for other human diseases.
In addition, the research shows that the compound, AN-3661, outperforms nitazoxanide, the only FDA approved treatment for Cryptosporidium, in cell lines and mouse models of the infection.
Beyond expanding the therapeutic options, that is important because the patients who are most severely affected by infestations of the intestinal parasite are also those for whom nitazoxanide does not work.
AN-3661 has been shown to be effective against other parasites such as Trypanosoma, Plasmodium and Toxoplasma. The two latter genera belong, with Cryptosporidium, to a phylum of parasites called apicomplexans, which are very close to each other from an evolutionary standpoint, said Christopher Swale, of the Institute of Advanced Sciences at the University of Grenoble, France.
“For this reason, we believed it interesting to directly test the efficacy [of AN-3661] on Cryptosporidium. Added to that [is] the fact that there is a real need for novel drugs against this parasite,” he told BioWorld.
Swale is lead author of a paper in the Nov. 6, 2019, issue of Science Translational Medicine, describing how the researchers succeeded in crystallizing the cleavage and polyadenylation specificity factor 3 (CPSF3), which is the target of AN-3661.
“Through this crystallization we managed to obtain the structure of the protein bound to AN-3661, a feat which had never been [achieved] for any of the other parasite organisms,” said Swale.
Doing this was “mostly luck,” Swale acknowledged. “Cryptosporidium is not a model organism, as it is actually one of the hardest to cultivate and genetically manipulate,” he said.
Exposing CPSF3 as the target of AN-3661 will enable the researchers to go on to rationally improve the binding of the drug and increase its specificity for parasitic CPSF3.
The protein is extremely well conserved from a sequential and structural viewpoint in all apicomplexan parasites and even in mammals. “This means that the understanding of the mechanism of action in Cryptosporidium will also serve drug development targeting CPSF3 for these other parasites as well,” said Swale.
In addition, the high-resolution structure will enable the researchers to look for structural elements which make the drug specific to parasites and non-toxic to mammals. As a target for de novo drug discovery, the applications may go beyond antiparasitic treatments, to human diseases where mRNA cleavage and polyadenylation is deregulated.
The substantial reduction in parasite load seen when mice were dosed over five days led the researchers to test a single dose of AN-3661, three days post infection, one day before the first oocytes would be shed.
That blocked oocyte release, and in evaluating the lowest possible dose needed to achieve that, it was shown AN-3661 is effective at a 10-fold lower dose than nitazoxanide.
Based on the high level of conservation of the DNA sequences of the CPSF3 binding site across apicomplexans, the researchers assessed the docking of other oxaboroles into the site in other parasites, demonstrating it is a shared mechanism with other species.
They said their structural data could guide the optimization and development of other oxaborole derivatives, potentially opening the way to a pan-apicomplexan treatment.
“What we show is that AN-3661 works very efficiently in the mouse model against Cryptosporidium infection, so the next stage would of course be to test it in bigger mammals,” Swale said. There also are potential veterinary applications of AN-3661, as both Cryptosporidium and Toxoplasma gondii cause serious infections in livestock as well.
The main goal now is to apply the findings about the mechanism of action to discover more compounds targeting CPSF3 that are effective against Cryptosporidium.
There are also many unresolved questions concerning the role of CPSF3 and also the inhibition process in the context of the much larger multisubunit CPSF complex with which it actually functions. “So there is still a lot of work ahead,” said Swale.
AN-3661 originally was discovered by Anacor Pharmaceuticals Inc., of Palo Alto, Calif., in a collaboration with the Medicines for Malaria Venture that started in 2010. In preclinical studies, it has shown activity against the artemisinin-resistant strains of the most aggressive human malarial parasite, Plasmodium falciparum. The compound has been progressively optimized and the latest version, designated AN-13762, is in preclinical development as a treatment for malaria.