Screening methodology for the identification of inhibitors targeting the lipoyl-relay of parasitic protozoa

SCATTOLINI, A.; PIANESI, T.; MANSILLA M.C.

Mar del Plata

Congreso; Congreso de la Sociedad Argentina de Microbiología General (SAMIGE); 2023

Sociedad Argentina de Microbiología General

Plasmodium falciparum (Pf) is a parasitic protozoan that causes the most aggressive form of malaria. Although there are treatments for this disease, their effectiveness is gradually declining, which generates the need to elucidate new pharmacological targets. Lipoic acid (LA) is an organosulfur cofactor required for the functioning of multienzyme complexes involved in oxidative metabolism, as well as being a potent antioxidant. Gram-positive bacteria, yeasts, trypanosomes, and mammals use a pathway called lipoyl-relay for protein lipoylation, which uses an amidotransferase to transfer the lipoyl groups to the different E2 subunits of the dehydrogenase complexes. In Pf, the lipoylation of these complexes becomes essential for their survival, both in the hepatic and in the blood phase of the infection. The amidotransferase of Trypanosoma brucei (TbLipL) has recently been validated as a new therapeutic target, so we decided to identify the activity of the homologous protein in Pf, PfLipL2, annotated as a lipoyl-transferase, and evaluate its potential as a new pharmacological target. To this end, the PflipL2 gene, encoding the PF3D7_0923600 protein, was expressed under the control of a xylose-inducible promoter in mutants of B. subtilis deficient in different steps of the synthesis and uptake of LA. No functional complementation of lipM mutants was observed, indicating that it does not possess octanoyltransferase (OT) activity. Neither was it possible to restore the growth of the double mutant lipA-lplJ, unable to synthesize and bind LA, even in the presence of the cofactor, ruling out lipoate ligase activity. In contrast, complementation of the growth of a lipL mutant was observed. To detect potential inhibitors of protozoan amidotransferases through high throughput screening we designed a B. subtilis reporter strain, NM12. This strain lacks LipL and express the OT LipB of E. coli and either PfLipL2 or TbLipL under a xylose inducible promoter. We took advantage of the ability of BrO to inhibit TbLipL and, as we demonstrated in this work, also Pf amidotransferase, to test hypothetical inhibition conditions in the reporter strain. To the best of our knowledge, this is the first screening method that has has been used to successfully identify amidotransferase inhibitors. The genes encoding amidotransferases from different organisms could also be introduced into the B. subtilis NM12 strain, demonstrating the extended versatility of the methodology.