Antileishmanial Aminopyrazoles: Studies into Mechanisms and Stability of Experimental Drug Resistance

Abstract

Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development. Resistance selection was performed by successive exposure of Leishmania infantum promastigotes (in vitro) and intracellular amastigotes (both in vitro and in golden Syrian hamsters). The stability of the resistant phenotypes was assessed after passage in mice and Lutzomyia longipalpis sandflies. Whole-genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs), and somy changes. The potential role of efflux pumps (the MDR and MRP efflux pumps) in the development of resistance was assessed by coincubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine, and probenecid). Repeated drug exposure of amastigotes did not result in the emergence of drug resistance either in vitro or in vivo Selection at the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index, 5.8 to 24.5). This phenotype proved to be unstable after in vivo passage in mice and sandflies, suggesting that nonfixed alterations are responsible for the elevated resistance. In line with this, single nucleotide polymorphisms and indels identified by whole-genome sequencing could not be directly linked to the decreased drug susceptibility. Copy number variations were absent, whereas somy changes were detected, which may have accounted for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. The selection performed does not suggest the rapid development of resistance against aminopyrazoles in the field. Karyotype changes may confer elevated levels of resistance, but these do not seem to be stable in the vertebrate and invertebrate hosts. MDR/MRP efflux pumps are not likely to significantly impact the activity of the aminopyrazole leads.

Keywords: ABC transporters; Leishmania; aminopyrazoles; resistance.

FIG 1

Overview of the stepwise generation of extracellular promastigote lines resistant to two APs.

FIG 2

Comparison of in vitro intracellular amastigote (A and C) and extracellular promastigote (B and D) susceptibility to the selected AP series between the wild-type parent (LEM3323Cl₄) and the successfully cultured adapted clones of the DNDI-1044-selected (A and B) and DNDI-8012-selected (C and D) resistant lines. Results are expressed as the mean IC₅₀ ± SEM and are based on two independent replicates run in duplicate (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). For DNDI-8012-generated clones, only a few clones were selected for amastigote susceptibility assessment.

FIG 3

Comparison of the in vitro amastigote multiplication ratio between the wild-type parent (LEM3323Cl₄) and the selected resistant lines. Results are expressed as the mean amastigote multiplication ratio ± SEM and are based on one experiment run in sextuplet (*, P < 0.05).

FIG 4

Effect of coincubation of verapamil, cyclosporine, and probenecid with the aminopyrazoles for L. infantum (A and C) and L. donovani (B and D) in the intracellular amastigote (A and B) and extracellular promastigote (C and D) susceptibility assays. Results are based on two independent repeats run in duplicate and are expressed as the mean IC₅₀ ± standard mean of error (SEM) (*, P < 0.05).