Catalytic bioscavengers as countermeasures against organophosphate nerve agents

Highlights

• Pre-treatment with catalytic bioscavengers can prevent nerve agent intoxications.

• They may also be effectively used for post-exposure therapy in certain cases.

• Obtained from natural enzymes using directed evolution and computational design.

• Clinical application requires research of safety, stability, delivery and production.

Abstract

Recent years have seen an increasing number of incidence, in which organophosphate nerve agents (OPNAs) have been used against civilians with devastating outcomes. Current medical countermeasures against OPNA intoxications are aimed at mitigating their symptoms, but are unable to effectively prevent them. In addition, they may fail to prevent the onset of a cholinergic crisis in the brain and its secondary toxic manifestations. The need for improved medical countermeasures has led to the development of bioscavengers; proteins and enzymes that may prevent intoxication by binding and inactivating OPNAs before they can reach their target organs. Non-catalytic bioscavengers such as butyrylcholinesterase, can rapidly bind OPNA molecules in a stoichiometric and irreversible manner, but require the administration of large protein doses to prevent intoxication. Thus, many efforts have been made to develop catalytic bioscavengers that could rapidly detoxify OPNAs without being inactivated in the process. Such enzymes may provide effective prophylactic protection and improve post-exposure treatments using much lower protein doses. Here we review attempts to develop catalytic bioscavengers using molecular biology, directed evolution and enzyme engineering techniques; and natural or computationally designed enzymes. These include both stoichiometric scavengers and enzymes that can hydrolyze OPNAs with low catalytic efficiencies. We discuss the catalytic parameters of evolved and engineered enzymes and the results of in-vivo protection and post-exposure experiments performed using OPNAs and bioscavengers. Finally, we briefly address some of the challenges that need to be met in order to transition these enzymes into clinically approved drugs.

Introduction

Current medical countermeasures for treating organophosphate nerve agent (OPNA) intoxication include: atropine, oxime reactivators, and anticonvulsants. If applied in time, they can prevent lethality and mitigate the intoxication symptoms. However, they may fail to prevent a cholinergic crisis that would lead to loss of consciousness and permanent brain damage [[1], [2], [3]]. In addition, they are not suitable as preventive measures prior to intoxication, as these drugs produce severe side effects such as: CNS impairment, increased blood pressure and increased heart rate if administered prior to an OPNA intoxication [4]. In fact, apart from the stoichiometric bioscavenger human butyrylcholinesterase (HuBChE), purified from human blood, there is no available prophylactic treatment that can prevent OPNA intoxication and the onset of its symptoms. In recent years, catalytic bioscavengers have been proposed as the next generation of medical countermeasures that may allow efficient prophylactic protection from high doses of OPNAs using small doses of protein [5,6].

Catalytic bioscavengers are enzymes that can detoxify OPNAs by performing multiple cycles of OPNA binding and hydrolysis. They have a clear advantage over stoichiometric ones since unlike the latter, which inactivate OPNAs by binding to them irreversibly, their interaction with OPNAs results in reversible binding and rapid hydrolysis of the OPNAs. In principle, this should enable small amounts of catalytic scavenger to detoxify lethal doses of nerve agents in-vivo before the latter can inactivate acetylcholinesterase (AChE) at important physiological sites; and to afford protection from multiple OPNA exposures without being consumed. In contrast, the high molecular weight of stoichiometric scavengers such as butyrylcholinesterase (BChE) or AChE and the requirement for a one-to-one ratio of non-catalytic scavenger to OPNA molecule, imply that large protein doses (i.e. hundreds of mgs) are required to provide protection from OPNA intoxication using stoichiometric scavengers [7,8]. Use of high protein doses of BChE or AChE for protection can be costly [9], and may also increase the chances of adverse physiological reactions following their administration [10]. Thus, during the past decade an increasing number of efforts have been devoted to the development of catalytic bioscavengers [5,6].

Pseudo-catalytic bioscavengers are enzymes or combinations of enzymes and chemical reactivators that can jointly perform multiple cycles of binding and hydrolysis of organophosphates. The term usually refers to stoichiometric scavengers from the family of B-esterases, such as AChE, BChE or carboxylesterase (CaE) [11], complexed with an oxime reactivator in their active site. Following the interaction of the enzyme with an OP inhibitor, it may undergo two consecutive reactions: First, covalent binding of the OP to its catalytic serine residue. Second, hydrolysis and release of the inactivating OP [12]. The rates of spontaneous detachment of the inhibitor from the catalytic serine depend on the type of enzyme and OP, but are usually very slow for B-esterases interacting with OPNAs. Therefore, external nucleophiles (i.e. chemical reactivators) are required to turn B-esterases into pseudo-catalytic OP hydrolyzing systems [[13], [14], [15]].

There are several advantages to pseudo-catalytic bioscavengers: First, their protein component is normally present in the circulation (e.g. BChE [16] in human serum and AChE [17] on red blood cells). Second, they rapidly sequester all types of OPNAs [18] as the latter are designed as cholinesterase inhibitors in the first place. Lastly, their reactivating oximes can reach inhibited AChE in peripheral nerve tissues and in some cases, in the central nervous system (CNS) [[19], [20], [21]]. However, there are several disadvantages to currently available reactivators: They exhibit narrow OPNA specificities and enable effective reactivation of only a number of OPNAs per reactivator [22,23]. They become ineffective following aging of the OP-cholinesterase bond, which is a rapid process in the case of OPNAs such as soman [24]. They have short circulatory residence times, and need replenishing in cases of continuing OPNA exposures [14]. Finally, the rate of OPNA hydrolysis by oxime-mediated reactivation, even by the most effective combinations of oxime, cholinesterase and OP (e.g. cyclosarin-inhibited HuAChE reactivated with HLö7, k_r2 = 9.3 × 10⁴ [M⁻¹min⁻¹] [25]), is ∼2–3 orders of magnitude slower than the hydrolytic rate required to efficiently prevent intoxication (see bellow). Therefore, the current status of pseudo-catalytic scavengers, suggests they are not efficient enough to detoxify OPNAs and prevent the inhibition of AChE at peripheral and CNS sites.

A more effective way of preventing OPNA intoxication would be to employ a highly efficient, broad-spectrum catalytic bioscavenger, which could rapidly inactivate the toxic isomers of OPNAs in the circulation. In order to do so using a low enzyme treatment-dose (<1 mg/kg), theoretical models predict that catalytic bioscavengers must have a catalytic efficiency (k_cat/K_M) of ≥1 × 10⁷ [M⁻¹ min⁻¹] with the toxic isomers of OPNAs [26,27]. Using enzymes with higher catalytic efficiencies (i.e. (k_cat/K_M) of ≥5 × 10⁷ [M⁻¹ min⁻¹]) as prophylactics would detoxify 96% of the OP in the circulation in less than 10 s and provide sign-free protection from intoxication [5,28]. Unfortunately, OPNAs are xenobiotic compounds that serve as promiscuous substrates for natural OP hydrolyzing (OPH) enzymes and are hydrolyzed with low catalytic efficiencies [29,30]. In addition, most OPH enzymes isolated so far tend to bind and inactivate the less toxic isomers of OPNAs more efficiently than the toxic ones [31,32]. Therefore, in order to obtain catalytic bioscavengers that could be used as effective medical countermeasures for OPNA intoxication, there is a need to greatly enhance both the activity and selectivity of natural enzymes towards the toxic isomers of OPNAs. In recent years, directed evolution and protein engineering techniques have been used successfully to generate catalytic bioscavengers with such properties [[33], [34], [35], [36], [37], [38]]. Here we aim to discuss proteins that have been suggested as candidates for catalytic bioscavenging, their activities and the efforts made to increase their OPNA hydrolyzing capabilities.