Tatyana Belinskaya

In search of a catalytic bioscavenger for the prophylaxis of nerve agent toxicity

A novel approach for treating organophosphorus (OP) poisoning is the use of enzymes, both stoichiometric and catalytic, as bioscavengers to sequester these compounds in circulation before they reach their physiological targets. Human serum butyrylcholinesterase and a recombinant form of this enzyme produced in the milk of transgenic goats have completed Phase I clinical trials as stoichiometric bioscavengers for the protection of humans against OP nerve agents. However, a major limitation of the first generation bioscavenger is the 1:1 stoichiometry between the enzyme and the OP. Therefore, efforts are underway to develop the second generation catalytic bioscavenger, which will neutralize/hydrolyze multiple OP molecules. To avoid any complications related to adverse immune reactions, three enzymes from human (Hu) sources are being considered for development as catalytic bioscavengers: (1) prolidase; (2) paraoxonase 1 (PON1); (3) senescence marker protein-30 (SMP-30). Towards this effort, native or recombinant (r) forms of candidate catalytic bioscavengers were isolated and characterized for their ability to hydrolyze G-type nerve agents at concentrations of 10muM and 1mM. Results show that mammalian enzymes were significantly less efficient at hydrolyzing nerve agents as compared to bacterial organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA). Recombinant Hu prolidase was the most efficient and the only mammalian enzyme that hydrolyzed all four G-type nerve agents. On the other hand, both rHu PON1 and Mo SMP-30 showed 10-fold lower activity towards sarin compared to rHu prolidase and did not hydrolyze tabun. Based on these results, Hu prolidase appears to be the most promising candidate for further development: (1) it can be easily expressed in E. coli; (2) of the three candidate enzymes, it is the only enzyme that hydrolyzes all four G-type agents. Efforts to improve the catalytic efficiency of this enzyme towards OP nerve agents are underway.

Exploiting Protein Fluctuations at the Active-Site Gorge of Human Cholinesterases: Further Optimization of the Design Strategy to Develop Extremely Potent Inhibitors

Protein conformational fluctuations are critical for biological functions, although the relationship between protein motion and function has yet to be fully explored. By a thorough bioinformatics analysis of cholinesterases (ChEs), we identified specific hot spots, responsible for protein fluctuations and functions, and those active-site residues that play a role in modulating the cooperative network among the key substructures. This drew the optimization of our design strategy to discover potent and reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase (hAChE and hBuChE) that selectively interact with specific protein substructures. Accordingly, two tricyclic moieties differently spaced by functionalized linkers were investigated as molecular yardsticks to probe the finest interactions with specific hot spots in the hChE gorge. A number of SAR trends were identified, and the multisite inhibitors 3a and 3d were found to be the most potent inhibitors of hBuChE and hAChE known to date.

Tacrine based human cholinesterase inhibitors: Synthesis of peptidic-tethered derivatives and their effect on potency and selectivity

Tacrine based reversible inhibitors of cholinesterases (ChEIs) containing peptidic tethers were synthesized to interact with specific regions at the gorge level, and their potency was determined with human (h) acetylcholinesterase and butyrylcholinesterase. Analogues 3i,j and 3l,m were identified as promising hits and may pave the way for the development of a new series of tacrine based enzyme selective hChEIs.

Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase.

Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed β-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 was d-(+)-gluconic acid δ-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The K(m) values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected δ-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal-ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.

In vitro characterization of organophosphorus compound hydrolysis by native and recombinant human prolidase.

Human prolidase is a binuclear metalloenzyme, which can potentially function as a catalytic bioscavenger for organophosphorus (OP) nerve agents. Although the biochemical properties of native prolidase purified from human erythrocytes, liver, kidney, and fibroblast cells are well known, it is very poorly characterized with regard to its OP hydrolyzing activity. Also, the high cost of purification of large quantities of native enzyme limits its use as a bioscavenger. Thus, recombinant human prolidase with similar biochemical properties to those of native enzyme would be more suitable as a catalytic bioscavenger. In this study, we established an Escherichia coli expression system, which produced a large amount of tagged human liver prolidase that was purified to over 95% purity from the soluble fraction of cell lysate by affinity chromatography on Streptavidin-agarose resin. The catalytic properties of the recombinant enzyme were compared in vitro with those of highly purified prolidase I isolated from human erythrocytes. The catalytic properties of recombinant prolidase overlap with those of the erythrocyte-derived native enzyme. Both enzymes efficiently hydrolyzed diisopropylfluorophosphate, sarin, soman, tabun and cyclosarin, but were much less efficient at hydrolyzing paraoxon and methyl paraoxon. These results suggest that human prolidase expressed in E. coli is suitable for further development as a catalytic bioscavenger for OP nerve agents.

Tetramer organizing polyproline-rich peptides identified by mass spectrometry after release of the peptides from Hupresin-purified butyrylcholinesterase tetramers isolated from milk of domestic pig (Sus scrofa)

Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10–12 M. The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the polyproline-rich peptides identified in PoBChE tetramers.

Characterization of butyrylcholinesterase from porcine milk

Human butyrylcholinesterase (HuBChE) is under development for use as a pretreatment antidote against nerve agent toxicity. Animals are used to evaluate the efficacy of HuBChE for protection against organophosphorus nerve agents. Pharmacokinetic studies of HuBChE in minipigs showed a mean residence time of 267 h, similar to the half-life of HuBChE in humans, suggesting a high degree of similarity between BChE from 2 sources. Our aim was to compare the biochemical properties of PoBChE purified from porcine milk to HuBChE purified from human plasma. PoBChE hydrolyzed acetylthiocholine slightly faster than butyrylthiocholine, but was sensitive to BChE-specific inhibitors. PoBChE was 50-fold less sensitive to inhibition by DFP than HuBChE and 5-fold slower to reactivate in the presence of 2-PAM. The amino acid sequence of PoBChE determined by liquid chromatography tandem mass spectrometry was 91% identical to HuBChE. Monoclonal antibodies 11D8, mAb2, and 3E8 (HAH 002) recognized both PoBChE and HuBChE. Assembly of 4 identical subunits into tetramers occurred by noncovalent interaction with polyproline-rich peptides in PoBChE as well as in HuBChE, though the set of polyproline-rich peptides in milk-derived PoBChE was different from the set in plasma-derived HuBChE tetramers. It was concluded that the esterase isolated from porcine milk is PoBChE.