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

• Published in: Angewandte Chemie International Edition 2021-07, Vol.60 (28), p.15613-15621
• Main Authors: Brand, Michael, Wang, Lei, Agnello, Stefano, Gazzola, Silvia, Gall, Flavio M., Raguž, Luka, Kaiser, Marcel, Schmidt, Remo S., Ritschl, Amélie, Jelk, Jennifer, Hemphill, Andrew, Mäser, Pascal, Bütikofer, Peter, Adams, Michael, Riedl, Rainer
• Format: Article
• Language: English
• Subjects: Antimicrobial Cationic Peptides - chemical synthesis; Antimicrobial Cationic Peptides - chemistry; Antimicrobial Cationic Peptides - pharmacology; antiparasitic agent; Antiprotozoal Agents - chemical synthesis; Antiprotozoal Agents - chemistry; Antiprotozoal Agents - pharmacology; Destabilization; drug discovery; Electron microscopy; Genetic screening; Leucinostatin; medicinal chemistry; Membrane potential; Membranes; Mimicry; Mitochondria; Mode of action; Model testing; Molecular Conformation; Parasitic Sensitivity Tests; peptides; Phospholipids; siRNA; Structure-Activity Relationship; Trypanosoma brucei brucei - drug effects; Trypanosoma brucei brucei - genetics; Water pollution effects
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202102153

Leucinostatin A is one of the most potent antiprotozoal compounds ever described, but little was known on structure–activity relationships (SAR). We used Trypanosoma brucei as a protozoal model organism to test synthetically modified derivatives, resulting 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. The antiprotozoal SAR matched SAR in phospholipid liposomes, where membrane integrity, leaking, and dynamics were studied. The mode of action is discussed based on a structure–activity analysis of derivatives in efficacy, ultrastructural studies in T. brucei, and artificial membrane models, mimicking 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, as demonstrated by ultrastructural analysis, electron microscopy and mitochondrial staining. Long‐time sublethal exposure of T. brucei (200 passages) and siRNA screening of 12′000 mutants showed no signs of resistance development to the synthetic derivatives. A structure–activity relationship of the natural product Leucinostatin A was performed to result in two highly potent compounds against the protozoa Trypanosoma brucei with decreased toxic effects in vitro and in vivo. Those compounds did not form any resistance by long‐time sublethal exposure and their mode of action lies in the destabilization of the inner mitochondrial membrane.