Plasmodium knowlesi exhibits distinct in vitro drug susceptibility profiles from those of Plasmodium falciparum

Abstract

New antimalarial agents are identified and developed after extensive testing on Plasmodium falciparum parasites that can be grown in vitro . These susceptibility studies are important to inform lead optimisation and support further drug development. Until recently, little was known about the susceptibility of non-falciparum species as these had not been adapted to in vitro culture. The recent culture adaptation of P . knowlesi has therefore offered an opportunity to routinely define the drug susceptibility of this species, which is phylogenetically closer to all other human malarias than is P . falciparum. We compared the in vitro susceptibility of P . knowlesi and P . falciparum to a range of established and novel antimalarial agents under identical assay conditions. We demonstrated that P . knowlesi is significantly less susceptible than P . falciparum to six of the compounds tested; and notably these include three ATP4 inhibitors currently under development as novel antimalarial agents, and one investigational antimalarial, AN13762, which is 67 fold less effective against P . knowlesi . For the other compounds there was a less than two-fold difference in susceptibility between species. We then compared the susceptibility of a recent P . knowlesi isolate, UM01, to that of the well-established, older A1-H.1 clone. This recent isolate showed similar in vitro drug susceptibility to the A1-H.1 clone, supporting the ongoing use of the better characterised clone to further study drug susceptibility. Lastly, we used isobologram analysis to explore the interaction of a selection of drug combinations and showed similar drug interactions across species. The species differences in drug susceptibility reported by us here and previously, support adding in vitro drug screens against P . knowlesi to those using P . falciparum strains to inform new drug discovery and lead optimisation.

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization of Plasmodium falciparum and Plasmodium knowlesi asexual stage parasites

“…The EC 50 for P. knowlesi was found to be higher than that for P. falciparum , even when P. falciparum was grown in the presence of serum, indicating a difference in biology that may make it slightly less susceptible to PKG inhibition. This is similar to previous findings, which established that the EC 50 values of certain anti-malarials are higher for P. knowlesi than for P. falciparum [51, 52]. Nonetheless, we have been able to successfully calibrate concentrations that are effective in the two species and in several media formulations (Table 1).…”

Novel Endochin-Like Quinolones Exhibit Potent In Vitro Activity against Plasmodium knowlesi but Do Not Synergize with Proguanil

“…We expect natural variability within our EC 50 values as our assays are run using asynchronous parasite populations, and because the parasites have different life cycle lengths, meaning drugs are exposed longer to P. falciparum per life cycle than for P. knowlesi. Hence, we only consider a greater than three-fold change in EC 50 between species as a potentially important species difference (19).…”

“…However, the activity of ELQs against the zoonotic P. knowlesi, an increasingly important cause of human malaria in Southeast Asia, is unknown. Importantly, recent articles have identified differences in in vitro (19,20) and ex vivo (21) susceptibility between P. knowlesi and P.…”

“…falciparum to established and experimental antimalarial agents. In particular, in vitro studies demonstrated that P. knowlesi is up to 8-fold less susceptible than P. falciparum to inhibitors of dihydroorotate dehydrogenase (e.g., DMS265 (20)), 6-fold less susceptible to ATP4 inhibitors (e.g., cipargamin, SJ733 (19)), around 3-fold less susceptible to cladosporin and pentamidine, and 66-fold less susceptible to the oxaborole AN13762 (19).…”

“…Conversely, P. knowlesi was shown to be 10-fold more susceptible to dihydrofolate reductase inhibitors (e.g., pyrimethamine, cycloguanil (20)), around 4.5-fold more susceptible to ganaplacide (KAF156), and over 3-fold more susceptible to halofantrine (19). In spite of the reduced susceptibility of P. knowlesi compared with P. falciparum, many antimalarials remain potent against P. knowlesi in vitro (e.g., 6 nM for cipargamin), and any clinical significance of these reported species differences is yet to be established.…”