The malaria parasite’s shifting defenses against antimalarial drugs have been shadowed by scientists on the lookout for new druggable targets. The scientists, led by researchers based at the University of California San Diego School of Medicine, used experimental evolution and whole-genome analysis to identify drug-resistance genes. The scientists conducted this work systematically, producing a map of the chemogenetic landscape that could guide the design of small-molecule inhibitors against the malaria parasite, which kills hundreds of thousands of people each year.
Details of the work appeared January 12 in the journal Science, in an article entitled “Mapping the Malaria Parasite Druggable Genome by Using In Vitro Evolution and Chemogenomics.” This article describes how the scientists performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. P. falciparum, a unicellular protozoan transmitted to humans through the bite of infected Anopheles mosquitos, is responsible for approximately half of all malaria cases.
“We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events,” wrote the article’s authors. “Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target–inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea.”
In 83 key genes that are associated with drug resistance, the researchers identified hundreds of changes that could be mediating this effect, including repeated genetic coding or mutations resulting in altered proteins. The researchers then used P. falciparum clones and exposed them to the compounds over time to induce resistance, monitoring the genetic changes that occurred as resistance developed.
Remarkably, the researchers found that they could identify a likely target, or resistance gene, for every compound. In particular, they identified mutations that repeatedly occurred upon individual exposure to a variety of drugs, meaning that these particular mutations are likely mediating resistance to numerous existing treatments.
“A single human infection can result in a person containing upwards of a trillion asexual blood stage parasites,” said Elizabeth Winzeler, Ph.D., the study’s senior author and professor of pharmacology and drug discovery in the department of pediatrics at UC San Diego School of Medicine. “Even with a relatively slow random mutation rate, these numbers confer extraordinary adaptability. In just a few cycles of replication, the P. falciparum genome can acquire a random genetic change that may render at least one parasite resistant to the activity of a drug or human-encoded antibody.”
Such rapid evolution poses significant challenges to controlling the disease, said researchers, but it can also be exploited in vitro to document precisely how the parasite evolves in the presence of known antimalarials to create drug resistance. It can also be used to reveal new drug targets.
Rather than focus upon the interaction of parasites to single compounds or investigate single suspect genes in P. falciparum, Dr. Winzeler and colleagues used whole-genome sequencing and a diverse set of antimalarial compounds. The resulting dataset revealed a diversity of mutations. Resistant parasites often contained a mutation in a presumptive target gene and additional mutations in other, unrelated genes.
This work confirmed previously known genetic modifications that substantially contribute to the parasites' drug resistance, but it also revealed new targets that deepen understanding of the parasites' underlying biology.
“Our findings showed and underscored the challenging complexity of evolved drug resistance in P. falciparum,” stated Dr. Winzeler, “but they also identified new drug targets or resistance genes for every compound for which resistant parasites were generated. It revealed the complicated chemogenetic landscape of P. falciparum, but also provided a potential guide for designing new small-molecule inhibitors to fight this pathogen.”