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

Abstract

Introduction: 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. Methodology: 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 resistant phenotypes was assessed after passage in mice and Lutzomyia longipalpis sand flies. Whole genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs) and somy changes. The potential role of efflux pumps (MDR/MRP) in the development of resistance was assessed by co-incubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine A and probenecid). Results: Repeated drug exposure of amastigotes did not result in the emergence of drug resistance, either in vitro or in vivo. Selection on the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index: 5-24). This phenotype proved to be unstable after in vivo passage in mice and sand flies, suggesting that non-fixed 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 account for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. Conclusion: The selection performed does not suggest 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.

Identification of Resistance Determinants for a Promising Antileishmanial Oxaborole Series

“…Although successful for other compounds, in vitro and in vivo resistance selection procedures were unable to generate Leishmania clones resistant to our oxaborole compounds. Additionally, the role of efflux pumps as a potential mechanism of resistance to these compounds was evaluated using inhibitors of ABC-transporters such as verapamil, cyclosporine A and probenecid [ 51 , 55 , 56 , 57 , 58 ]. These studies suggest that antileishmanial oxaboroles are not substrates of the evaluated transporters, unlike several other antileishmanials shown to be prone to efflux through these pumps [ 39 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 ].…”

“…The medium was replaced 2 h later with fresh RPMI-1640 medium containing oxaborole and one of the ABC-transporter inhibitors, namely, verapamil (MDR and MRP inhibitor), cyclosporine A (broad specificity efflux inhibitor), or probenecid (MRP inhibitor). The ABC-transporter inhibitor was added at a single concentration below its EC 50 which was previously determined [ 51 ]; verapamil was added at 8 µM, probenecid at 700 µM, and cyclosporine A at 1.5 µM for L. infantum and 2 µM for L. donovani . A 4-fold dilution series was prepared for the oxaboroles with the highest in-test concentration of 10 µM.…”

“…L. infantum and L. donovani log-phase promastigotes were diluted to a concentration of 10 6 promastigotes/well in a 96-well plate, whereafter dilutions of Sb III and oxaborole were added either with or without the ABC-transporter inhibitor. Pump inhibitor concentrations were previously determined and were identical to those in the intracellular assay [ 51 ]. EC 50 -values with and without ABC-transporter inhibitor were determined as described above in the extracellular susceptibility assay and the different conditions were compared.…”

Road‐map of pre‐clinical treatment for Visceral Leishmaniasis

“…In a recent study, the EC 50 and EC 90 values of the four different drugs (AmB, miltefosine, paramomycin and SSG) under different conditions of media flow like static, low flow and high flow were evaluated. Besides, IC 50 values of three different drugs DNDi‐1044, DNDi‐8012 and DNDi‐5561 were also evaluated using intracellular and extracellular promastigote assays (Van den Kerkhof et al, 2020). The involvement of efflux pump inhibitors like MDR and MRP were evaluated.…”

“…AmB, miltefosine, paramomycin and SSG) under different conditions of media flow like static, low flow and high flow were evaluated.Besides, IC 50 values of three different drugs DNDi-1044, DNDi-8012 and DNDi-5561 were also evaluated using intracellular and extracellular promastigote assays (Van denKerkhof et al, 2020). The involvement of efflux pump inhibitors like MDR and MRP were evaluated.The ABC transport inhibitors were evaluated for their cytotoxic effects on MRC5 cells and their inhibitors effect on both amastigote and promastigote forms (Van denKerkhof et al, 2020). To determine the drug susceptibility of clinical Leishmania isolates in laboratory conditions, different cell-based drug susceptibility assays were evaluated.…”