New antimalarial drugs are needed. The benzoxaborole AN13762 showed excellent activity against cultured Plasmodium falciparum, against fresh Ugandan P. falciparum isolates, and in murine malaria models. To gain mechanistic insights, we selected in vitro for P. falciparum isolates resistant to AN13762. In all of 11 independent selections with 100 to 200 nM AN13762, the 50% inhibitory concentration (IC50) increased from 18–118 nM to 180–890 nM, and whole-genome sequencing of resistant parasites demonstrated mutations in prodrug activation and resistance esterase (PfPARE). The introduction of PfPARE mutations led to a similar level of resistance, and recombinant PfPARE hydrolyzed AN13762 to the benzoxaborole AN10248, which has activity similar to that of AN13762 but for which selection of resistance was not readily achieved. Parasites further selected with micromolar concentrations of AN13762 developed higher-level resistance (IC50, 1.9 to 5.0 μM), and sequencing revealed additional mutations in any of 5 genes, 4 of which were associated with ubiquitination/sumoylation enzyme cascades; the introduction of one of these mutations, in SUMO-activating enzyme subunit 2, led to a similar level of resistance. The other gene mutated in highly resistant parasites encodes the P. falciparum cleavage and specificity factor homolog PfCPSF3, previously identified as the antimalarial target of another benzoxaborole. Parasites selected for resistance to AN13762 were cross-resistant with a close analog, AN13956, but not with standard antimalarials, AN10248, or other benzoxaboroles known to have different P. falciparum targets. Thus, AN13762 appears to have a novel mechanism of antimalarial action and multiple mechanisms of resistance, including loss of function of PfPARE preventing activation to AN10248, followed by alterations in ubiquitination/sumoylation pathways or PfCPSF3. IMPORTANCE Benzoxaboroles are under study as potential new drugs to treat malaria. One benzoxaborole, AN13762, has potent activity and promising features, but its mechanisms of action and resistance are unknown. To gain insights into these mechanisms, we cultured malaria parasites with nonlethal concentrations of AN13762 and generated parasites with varied levels of resistance. Parasites with low-level resistance had mutations in PfPARE, which processes AN13762 into an active metabolite; PfPARE mutations prevented this processing. Parasites with high-level resistance had mutations in any of a number of enzymes, mostly those involved in stress responses. Parasites selected for AN13762 resistance were not resistant to other antimalarials, suggesting novel mechanisms of action and resistance for AN13762, a valuable feature for a new class of antimalarial drugs.

The HIV protease inhibitor lopinavir inhibits Plasmodium falciparum aspartic proteases (plasmepsins) and parasite development, and children receiving lopinavir-ritonavir experienced fewer episodes of malaria than those receiving other antiretroviral regimens. Resistance to lopinavir was selected in vitro over ϳ9 months, with ϳ4-fold decreased sensitivity. Whole-genome sequencing of resistant parasites showed a mutation and increased copy number in pfmdr1 and a mutation in a protein of unknown function, but no polymorphisms in plasmepsin genes.KEYWORDS malaria, Plasmodium falciparum, drug sensitivity, drug resistance, pfmdr1, aspartic protease, antiretroviral, lopinavir, HIV, drug resistance mechanisms I nfection with Plasmodium falciparum, the most virulent human malaria parasite, causes hundreds of millions of illnesses and hundreds of thousands of deaths each year (1). Despite recent progress in some regions, the treatment and control of the disease are challenged by increasing resistance to available therapies (2). New drugs to treat malaria are needed. One approach is to repurpose drugs now used for other indications to treat or prevent malaria.The P. falciparum genome sequence predicts the presence of 10 aspartic proteases, known as plasmepsins (3). Plasmepsins I, II, III (also known as histo-aspartic protease), and IV hydrolyze hemoglobin in the plasmodial food vacuole, in concert with other proteases, to provide amino acids for erythrocytic parasites (4). Plasmepsin V is an endoplasmic reticulum protease that cleaves proteins bound for export to the erythrocyte (5, 6). The functions of plasmepsins VI to X are unknown, with different enzymes believed to be active in erythrocyte-and mosquito-stage parasites (7,8).The HIV protease is also an aspartic protease (9), and inhibitors of this enzyme are among our most important antiretroviral drugs (10). A number of antiretroviral protease inhibitors have been shown to inhibit plasmepsins (11), to be active against cultured malaria parasites (11, 12), and to effectively treat murine malaria (13). Lopinavir, which is used to treat HIV in combination with ritonavir, is active against P. falciparum at low micromolar concentrations that are below the levels achieved by standard dosing (11). HIV-infected Ugandan children who received lopinavir/ritonavir had decreased incidence of malaria compared to those receiving a regimen that did not include a protease inhibitor (14). The impact of lopinavir/ritonavir appeared to be mediated principally by prolonged exposure to the antimalarial lumefantrine after therapy, due to inhibition of metabolism by ritonavir, rather than by protease inhibition, as the effect was greatest in episodes following prior therapy with artemetherlumefantrine. However, considering only the first episodes of malaria, and thus remov- Citation Sonoiki E, Nsanzabana C, Legac J, Sindhe KMV, DeRisi J, Rosenthal PJ. 2017. Altered Plasmodium falciparum sensitivity to the antiretroviral protease inhibitor lopinavir associated with polymorphisms in pf...

The Early Transcribed Membrane Proteins (ETRAMPs) belong to a multigene family which are conserved, are specific to Plasmodium species, and abundantly present in parasitophorous vacuolar membrane (PVM). The functions of the members of this family are poorly understood. PfETRAMP14.1 (PF3D7_1401400) is a member of this family, present only in Plasmodium falciparum. In this study, we report the potential interacting partners of PfETRAMP14.1 by using immunoprecipitation (IP) LC-MS/MS as well as protein-interaction network reconstructed on in vivo array analyses of severe malaria inflicted patients from malaria endemic Indian regions. We find PfETRAMP14.1 to be the most highly transcribed gene in severe infection. Our studies with western blot analysis and Immuno-flurorescence show that PfETRAMP14.1 is expressed at PVM during all the intraerythrocytic stages of P.falciparum with maximum expression at early trophozoite stage. Further, our results reveal interactions of ETRAMP14.1 with Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1), thioredoxin (TRX2), export protein 2 (EXP2), heat shock protein 70-1 (Hsp70-1) and some of the ETRAMP family members. We propose that ETRAMP14.1 helps trafficking of PfEMP1 to the host RBC surface in conjunction with translocon machinery and the chaperone HSP 70-1.

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