Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun.
hBChE [human BChE (butyrylcholinesterase)] naturally scavenges OPs (organophosphates). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated ChEs (cholinesterases) can undergo a spontaneous time-dependent process called 'aging' during which the conjugate is dealkylated, leading to creation of an enzyme that cannot be reactivated. hBChE inhibited by phosphoramidates such as tabun displays a peculiar resistance to oxime-mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates by determining the kinetics of inhibition, reactivation (obidoxime {1,1'-(oxybis-methylene) bis[4-(hydroxyimino) methyl] pyridinium dichloride}, TMB-4 [1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide], HLö 7 {1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate)}, HI-6 {1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate} and aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to a previous observation made on tabun-ChE conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that highlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides the structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment, or to design BChE-based catalytic bioscavengers.
Carletti
Eugénie
E
Unité d'Enzymologie, Département de Toxicologie, Centre de Recherches du Service de Santé des Armées (CRSSA), 24 av des Maquis du Grésivaudan, 38700 La Tronche, France.
Aurbek
Nadine
N
Gillon
Emilie
E
Loiodice
Mélanie
M
Nicolet
Yvain
Y
Fontecilla-Camps
Juan-Carlos
JC
Masson
Patrick
P
Thiermann
Horst
H
Nachon
Florian
F
Worek
Franz
F
D002091:Butyrylcholinesterase / Q000378:metabolism*; D020134:Catalytic Domain; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D002801:Cholinesterase Reactivators / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D010755:Organophosphates / Q000494:pharmacology*; D010091:Oximes / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013997:Time Factors
D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D010755:Organophosphates; D010091:Oximes; D002091:Butyrylcholinesterase; C009374:tabun