Irreversible Inactivation of Trypanothione Reductase by Unsaturated Mannich Bases:  A Divinyl Ketone as Key Intermediate

• Published in: Journal of medicinal chemistry Vol. 48; no. 23; pp. 7400 - 7410
• Main Authors: Lee, Brittany, Bauer, Holger, Melchers, Johannes, Ruppert, Thomas, Rattray, Lauren, Yardley, Vanessa, Davioud-Charvet, Elisabeth, Krauth-Siegel, R. Luise
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 17.11.2005; Amer Chemical Soc
• Subjects: Animals; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antimalarials - chemistry; Antimalarials - pharmacology; Antiparasitic agents; Antiprotozoal Agents - chemistry; Antiprotozoal Agents - pharmacology; Biological and medical sciences; Chemistry, Medicinal; Chromatography, High Pressure Liquid; Dimethyl Sulfoxide - chemistry; Drug Storage; Glutathione - chemistry; Glutathione Reductase - chemistry; Humans; Ketones - chemistry; Leishmania donovani - drug effects; Life Sciences & Biomedicine; Magnetic Resonance Spectroscopy; Mannich Bases - chemistry; Mannich Bases - pharmacology; Medical sciences; NADH, NADPH Oxidoreductases - antagonists & inhibitors; NADH, NADPH Oxidoreductases - chemistry; Pharmacology & Pharmacy; Pharmacology. Drug treatments; Plasmodium falciparum - drug effects; Science & Technology; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thioredoxin-Disulfide Reductase - chemistry; Trypanocidal Agents - chemistry; Trypanocidal Agents - pharmacology; Trypanosoma brucei rhodesiense - drug effects; Trypanosoma cruzi - drug effects; GLUTATHIONE-REDUCTASE; LIPOAMIDE DEHYDROGENASE; ENZYME; DROSOPHILA-MELANOGASTER; ACTIVE-SITE; TRYPANOSOMA-CRUZI; PLASMODIUM-FALCIPARUM; THIOREDOXIN REDUCTASE; LEISHMANIA-DONOVANI; CONJUGATED STYRYL KETONES; Kinetoplastida; Protozoa; Trypanosoma; Enzyme; Enzyme inhibitor; Ketone; In vitro; Biological activity; Antiprotozoal agent; Chlorine Organic compounds; Parasiticide; Oxidoreductases; Mechanism of action; Chemical synthesis; Enone; Trypanothione reductase
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/jm0504860

Trypanothione reductase is a flavoenzyme unique to trypanosomatid parasites. Here we show that unsaturated Mannich bases irreversibly inactivate trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The inhibitory potency of the compounds strongly increased upon storage of the DMSO stock solutions. HPLC, NMR, and mass spectrometry data of potential intermediates revealed a divinyl ketone as the active compound inactivating the enzyme. ESI- and MALDI-TOF mass spectrometry of trypanothione reductase modified by the Mannich base or the divinyl ketone showed specific alkylation of the active site Cys52 by a 5-(2‘chlorophenyl)-3-oxo-4-pentenyl substituent. The reaction mechanism and the site of alkylation differ from those in Plasmodium falciparum thioredoxin reductase where the C-terminal redox active dithiol is modified. After deamination, unsaturated Mannich bases are highly reactive in polycondensation with trypanothione. Interaction of these compounds with both trypanothione and trypanothione reductase could account for their potent trypanocidal effect against Trypanosoma brucei.