Plasmodium falciparum Resistance to a Lead Benzoxaborole Due to Blocked Compound Activation and Altered Ubiquitination or Sumoylation

Abstract

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.

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs

“…brucei aldehyde dehydrogenase [ 24 ]. More recently, the benzoxaborole AN13762 was found to be intracellularly hydrolysed in Plasmodium falciparum by a lysophospolipase homologue, whose loss of function was linked to resistance [ 25 ].…”

Target Identification of an Antimalarial Oxaborole Identifies AN13762 as an Alternative Chemotype for Targeting CPSF3 in Apicomplexan Parasites

“…Although it is clear that AN13762 targets CPSF3 in T. gondii , different results were observed in P. falciparum where the mechanism of resistance is plural ( Sindhe et al., 2020 ). In fact, while we were investigating the mechanism of action of AN13762 in T. gondii , Sindhe and colleagues have shown that P. falciparum resistance depends not only on the activity of Prodrug Activation and Resistance Esterase ( Pf PARE), an enzyme responsible for AN13762 processing, but also on enzymes involved in ubiquitination and SUMOylation pathways or Pf CPSF3.…”

“…Note that no mutations with significant enrichment were found in TGGT1_306330, the closest homolog to Pf PARE in T. gondii ( Table S2 ). Furthermore, as AN13762 processing is required for full antimalarial activity, it is tempting to speculate that the lack of intracellular activation explains the decreased sensitivity observed in T. gondii and Cryptosporidium (EC 50 values are in the μM range, Figures 1 C and 5 B) relative to P. falciparum (EC 50 values ranging from 18 to 118 nM, Sindhe et al., 2020 ).…”

“…AN13762 has not exhibited either significant toxicology or cytotoxicity liabilities at any dose tested, and AN13762 was selected for preclinical development by Medicines for Malaria Venture in 2017 ( Zhang et al., 2017 ). Although previous research on parental scaffold AN3661 identified Cleavage and Polyadenylation Specificity Factor 3 (CPSF3) as the direct target ( Palencia et al., 2017 ; Sonoiki et al., 2017 ; Swale et al., 2019 ), a recent study investigating the resistance mechanisms of AN13762 in P. falciparum identified multiple components involved in prodrug activation or sumoylation and ubiquitination pathways along with Pf CPSF3 suggesting that the latter was not the primary target ( Sindhe et al., 2020 ).…”

Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites

“…The presence of multiple amino acid residues on Pf Uba2 and Pf Ubc9 that dictate the selectivity of their interaction indicates potential for development of parasite-specific inhibitors targeting this pathway [ 62 , 63 ]. Furthermore, mutations in both Pf Uba2 and the Pf SUMO E3 ligase PIAS arose during selection for resistance to high concentrations of the benzoxaborole drug AN13762, underscoring the pertinence of the entire SUMO pathway to the mode of action of this drug and potentially to other antimalarials [ 67 ]. Considering that Pf SUMO and the associated enzymatic machinery are essential for survival of P. falciparum [ 57 ], selective disruption of the key steps in the attachment of this Ubl to substrates is likely to be lethal to malaria parasites.…”