In vitro evaluation of the catalytic activity of paraoxonases and phosphotriesterases predicts the enzyme circulatory levels required for in vivo protection against organophosphate intoxications

Abstract

Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme]pl), established prior to the OP challenge, with the severity of symptoms and survival rates of intoxicated animals. The major objective was to apply a theoretical approach to estimate a lower limit for (kcat/KM)[Enzyme]pl that will be adequate for establishing the desired kcat/KM value and plasma concentration of efficacious catalytic bioscavengers. Published data sets by our group and others, from in vivo protection experiments executed in the absence of any supportive medicine, were analyzed. The kcat/KM values of eight OP hydrolyzing enzymes and their plasma concentrations in four species exposed to OPs via s.c., i.m. and oral gavage, were analyzed. Our results show that regardless of the OP type and the animal species employed, sign-free animals were observed following bioscavenger treatment provided the theoretically estimated time period required to detoxify 96% of the OP (t96%) in-vivo was ≤ 10 s. This, for example, can be achieved by an enzyme with kcat/KM = 5×107 M-1min-1 and a plasma concentration of 0.4 μM ((kcat/KM)[Enzyme]pl=20 min-1). Experiments in which animals were intoxicated by i.v. OP injections did not always conform to this rule, and in some cases resulted in high mortality rates. We suggest that in vivo evaluation of catalytic scavengers should avoid the unrealistic bolus i.v. route of OP exposure.

Cholinesterase reactivators and bioscavengers for pre‐ and post‐exposure treatments of organophosphorus poisoning

“…; Ashani et al . ). Directed evolution of a chimeric PON‐1 made via gene shuffling, combined with high‐throughput screening, successfully led to very active evolved variants.…”

“…; Ashani et al . ). But no PON‐1 mutants capable of hydrolyzing V‐agents at high rates have been found so far, thus preventing the development of broad‐spectrum bioscavengers.…”

Counteracting poisoning with chemical warfare nerve agents

“…Catalytic bioscavengers are enzymes that can hydrolyse NAs into non-toxic products. The advantage of catalytic over stoichiometric bioscavengers is that they need lower doses to provide superior protection of native AChE from phosphylation, because one enzyme degrades multiple NAs molecules ( 207 , 208 ). Several natural occurring human enzymes whose substrates are OPs are being investigated as potential catalytic bioscavengers, such as PON1 from plasma, erythrocyte and liver prolidase, carboxylesterase, platelet activating factor acetylhydrolase, liver senescence marker, and cytosolic aminopeptidases ( 142 , 144 , 209 , 210 , 211 , 212 ).…”

“…The greatest interes is for PON1 which hydrolyses several OP compounds at a high rate, it is enantioselective, prefers the more toxic S(–) enantiomer of tabun, but, unfortunately, shows greater enantioselectivity to the less toxic isomers of soman ( 213 , 214 ). Increasing PON1 catalytic activity up to 100 times would be sufficient to effectively scavenge various nerve agents, which is why scientists have been looking into mutations that would increase its catalytic activity ( 142 , 208 , 209 ). PON1 can be isolated from plasma but is complexed with HDL cholesterol, which renders isolation and purification expensive and complicated, and the isolated enzyme unstable ( 142 , 209 ).…”

Optimization of Cholinesterase-Based Catalytic Bioscavengers Against Organophosphorus Agents

“…If k cat / K m > 5 × 10 7 M −1 ·min −1 then the half time for complete hydrolysis of OP would be <1 s (Worek et al, 2016a ). To date, the most promising catalytic bioscavengers are evolved enantioselective phosphotriesterases (PTEs), such as evolved Brevundimonas ( Pseudomonas) diminuta PTE and evolved paraoxonase-1 (PON-1) (Ashani et al, 2016 ). The evolved mutant of B. diminuta PTE, L7ep-3, displays k cat / K m = 4.8 × 10 7 M −1 ·min −1 for S p VX and k cat / K m = 1.6 × 10 5 M −1 ·min −1 for S p VR (Bigley et al, 2015 ).…”