Irreversible inactivation of trypanothione reductase by unsaturated Mannich bases: a divinyl ketone as key intermediate.
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.
Lee
Brittany
B
Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 504, 69120 Heidelberg, Germany.
Bauer
Holger
H
Melchers
Johannes
J
Ruppert
Thomas
T
Rattray
Lauren
L
Yardley
Vanessa
V
Davioud-Charvet
Elisabeth
E
Krauth-Siegel
R Luise
RL
D000818:Animals; D000962:Antimalarials / Q000737:chemistry / Q000494:pharmacology; D000981:Antiprotozoal Agents / Q000737:chemistry* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004356:Drug Storage; D005978:Glutathione / Q000737:chemistry; D005980:Glutathione Reductase / Q000737:chemistry; D006801:Humans; D007659:Ketones / Q000737:chemistry*; D007893:Leishmania donovani / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy; D008352:Mannich Bases / Q000737:chemistry* / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D010963:Plasmodium falciparum / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry; D014344:Trypanocidal Agents / Q000737:chemistry / Q000494:pharmacology; D016833:Trypanosoma brucei rhodesiense / Q000187:drug effects; D014349:Trypanosoma cruzi / Q000187:drug effects
C507032:1-(2'-chlorophenyl)penta-1,4-dien-3-one; D000962:Antimalarials; D000981:Antiprotozoal Agents; D007659:Ketones; D008352:Mannich Bases; D014344:Trypanocidal Agents; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione; D004121:Dimethyl Sulfoxide