Antiprotozoal Structure–Activity Relationships of Synthetic Leucinostatin Derivatives and Elucidation of their Mode of Action

Abstract

Leucinostatin Ai soneo ft he most potent antiprotozoal compounds ever described, but little was knowno n structure-activity relationships (SAR). We used Trypanosoma brucei as ap rotozoal model organism to test synthetically modified derivatives,r esulting in simplified but equally active compounds 2 (ZHAWOC6025) and 4 (ZHAWOC6027), which were subsequently modified in all regions of the molecule to gain an in-depth SAR understanding.T he antiprotozoal SAR matched SAR in phospholipid liposomes, where membrane integrity,l eaking, and dynamics were studied. The mode of action is discussed based on as tructure-activity analysis of derivatives in efficacy,u ltrastructural studies in T. brucei, and artificial membrane models,m imicking membrane stability and membrane potential. The main site of antiprotozoal action of natural and synthetic leucinostatins lies in the destabilization of the inner mitochondrial membrane,a sd emonstrated by ultrastructural analysis,e lectron microscopya nd mitochondrial staining.L ong-time sublethal exposure of T. brucei (200 passages) and siRNAs creening of 12'000 mutants showed no signs of resistance development to the synthetic derivatives.

Differential Affinity Chromatography Coupled to Mass Spectrometry: A Suitable Tool to Identify Common Binding Proteins of a Broad-Range Antimicrobial Peptide Derived from Leucinostatin

“…Herein, we show that peptide 6027 is also highly effective against the apicomplexan parasite T. gondii , by inhibiting tachyzoite proliferation in vitro with an IC 50 in a similar range as that reported for T. b. rhodesiense bloodstream forms [ 17 ], most likely by hitting the mitochondrion as the main subcellular target affecting its structural integrity within a few hours of treatment, similar to what was previously observed with T. brucei bloodstream forms [ 17 ]. Thus, the destabilization of the inner mitochondrial membrane could be a common mechanism of action.…”

“…Cell culture media was purchased from Gibco-BRL (Zürich, Switzerland), and biochemical agents were procured from Sigma (St. Louis, MO, USA). The antimicrobial leucinostatin derivatives were synthesized at the Zürcher Hochschule für Angewandte Wissenschaft as described earlier [ 17 ].…”

“…Therefore, they constitute an ideal class of compounds for antimicrobial drug development. Recently, using Trypanosoma brucei (Alveolata) as a model organism, derivatives of the antibiotic peptide leucinostatin [ 16 ], have been identified as powerful antiprotozoal agents most likely affecting the stability of the inner mitochondrial membrane [ 17 ]. In the present study, we present the effects of one of the most promising compounds of this study, the peptide ZHAWOC_6027 (henceforth referred to as peptide 6027), on Toxoplasma gondii and mouse splenocytes.…”

Identification of Leucinostatins from Ophiocordyceps sp. as Antiparasitic Agents against Trypanosoma cruzi

“…Recent work with SAR using a leucinostatin A scaffold against T. brucei and Leishmania donovani has further demonstrated experimentally that the principal mechanism of action of leucinostatins is through destabilization of the inner mitochondrial membrane in these parasites …”

“…Recent work with SAR using a leucinostatin A scaffold against T. brucei and Leishmania donovani has further demonstrated experimentally that the principal mechanism of action of leucinostatins is through destabilization of the inner mitochondrial membrane in these parasites. 34 Leucinostatins are chemically tractable natural products for an SAR campaign, and the total synthesis of molecules in this class has been demonstrated. 35 Peptidic leads for drug discovery programs for Chagas disease have been explored in a number of other publications from our group and others.…”