Authors
D Oliveira2; D Oliveira3; D Oliveira1; P G Steel1; P Denny1; B ROSSI-BERGMANN2; B ROSSI-BERGMANN3; 1 Durham University; 2 UFRJ, Rio de Janeiro, RJ, Brazil; 3 UFRJ, Rio de Janeiro, RJ, Brazil Discussion
Chalcones are a promising new class of antileishmanials whose target is still unknown. In previous work, we have reported the selective activity of the chalcone CH8 against several Leishmania spp., suggesting a conserved drug target. In this work we synthesized CH8 analogues with alkyne groups for cycloaddition coupling (Huisgen-Sharpless reaction) without affecting antileishmanial activity. To discover the protein target of CH8 we then synthesized probes containing biotin (DEO53) or a fluorophore (PGS14.4), and an azide group allowing chemical linkage to the CH8 analogues. Two CH8 analogues, NAT22 and DEO37, and two probes, DEO53 (biotin-tagged) and PGS14.4 (rhodamine-tagged) were screened against L. amazonensis and L. major insect stage promastigotes. NAT22 and DE037 showed excellent activity against both species, IC50 0.3-2.2μM. However, the PGS14.4 and DEO53 probes had significantly decreased activity against L. amazonensis and L. major, probably due to their large molecular size. To allow these molecules work to reach the chalcone target(s), L .amazonensis promastigotes were permeated (digitonin), incubated with fluorescent PGS14.4 and imaged using confocal microscopy. The probe primarily localized to the mitochondria. L .amazonensis promastigotes pre-incubated with DEO37 were then lysed by sonication and a subsequent click reaction allowed the purification of bound proteins. In summary, these probes appear to be a useful tool for the identification of the chalcone target(s) in Leishmania spp. Supported by: CAPES, CNPq 
