Authors

Discussion

Compounds from the GSK Leishbox are active anti-leishmanials identified by phenotypic screening, but many of them have an unknown mode-of-action. This study aimed to identify the targets of a subset of nine LeishBox compounds, reduced to four following initial triage. To achieve this, drug-resistant strains of L. major and L. mexicana promastigotes were generated by in vitro evolution and then subjected to whole-genome sequencing. Following SNP calling and identification of coding mutations, four potential targets were selected for genetic validation, including two hypothetical proteins, one encoding a predicted amino acid transporter (AATP11), and one encoding a folate biopterin transporter (FBT).

 Initially, null mutants were generated in L.mx T7/Cas9 to separate likely essential targets from non-essential genes. Null mutants could be generated for all four genes, suggesting that these are not the sole targets of the compounds. Furthermore, since two of the proteins were predicted to be transporters, mutations in their genes were hypothesized to confer resistance to the compounds. When the ∆aatp11 strain was treated with compound 3, it resulted in a 2-fold increase in the EC₅₀ of the compound. Addback and over-expressor strains have been generated to confirm that this phenotype is gene-specific. The ∆fbt strain is currently being assayed. This target is part of a tandem gene array that is often mutated in response to anti-folates such as methotrexate.