Serum paraoxonase (PON1) is a calcium-dependent lactonase, with lipophilic lactones constituting its primary substrates [1
]. When associated with HDL, an increase in the stability and lipo-lactonase activity of PON1 were measured both in vivo
and in vitro
]. Also, HDL-PON1 complex inhibits LDL oxidation [6
], and stimulates cholesterol efflux from macrophages [8
]. Previous studies of PON1 showed that knockout mice were highly susceptible to atherosclerosis [9
], and serum PON1 levels, and polymorphism, were related to the level of cardiovascular disease [10
], all of which indicate a role of PON1 for the prevention of atherosclerosis. PON1 also exhibits hydrolytic activity against certain organophosphates (OPs), including the toxic oxon metabolites of a number of insecticides, and nerve agents such as sarin and soman [12
], and has thus the potential to protect against OP poisoning. Indeed, PON1 knockout mice exhibit a significant increase in sensitivity to diazoxon [14
], paraoxon, chlorpyrifos and chlorpyrifos-oxon [9
], and the toxic effects can be reversed by administrating rabbit PON1 [15
]. Although these properties render PON1 an attractive candidate for the treatment of atherosclerosis, and pesticides or nerve agents toxicity, certain characterizations of human PON1 hamper such uses.
Human PON1 (huPON1), is sensitive to a range of challenges, including the presence of oxidizing agents, glucose, and thiols [16
]. The complex of HDL (specifically apoA-I), stabilizes the enzyme. Thus, when anchored onto functional HDL-apoA-I, PON1 exhibits anti-atherogenic activity [20
], but not in its lipid-free form [21
]. However, cardiovascular disease (CVD) involves the modification of HDL composition and structure giving rise to "dysfunctional HDL" [23
]. HDL-associated enzymes including PON1 become dysfunctional and/or depleted under these conditions, as well as under inflammatory conditions [24
], and metabolic diseases such as type 1 and type 2 diabetes [23
], metabolic syndrome (MetS) [26
], and premature CVD [27
Acute-phase response is also associated with decreased PON1 activity, probably due to the displacement of PON1 from HDL [26
]. It appears, therefore, that a highly robust PON1, and perhaps a regeneration of HDL particles, might be needed for therapeutic applications, as demonstrated by the application of apoA-I Milano [28
] and apoA-I mimetics [29
]. The application of HDL-PON1 complex with improved stability and efficacy as described in this paper might therefore be needed for effective HDL-therapy.
In addition, the catalytic efficiency of huPON1 with most organophosphates, and effectively all highly toxic nerve agents, is not sufficiently high to provide substantial protection [14
]. In fact, PON1's activity with many OPs is comparable to the weak, promiscuous activity of serum albumin towards these agents [31
]. Another limitation of huPON1 is its poor stability and tendency for aggregation [32
]. This may limit the therapeutic usages of the enzyme in which relatively high concentrations are administered by the intravenous route.
Directed evolution is extensively used to improve protein properties, such as stability, binding affinity, or catalytic efficiency. We have applied directed evolution to generate recombinant PON1 (rePON1) that expresses in a soluble and functional form in E. coli
, and exhibits enzymatic properties, and HDL binding and stimulation capabilities, that are essentially identical to those of huPON1 [34
]. The often-used rePON1 variant G3C9 is closest in sequence to rabbit PON1 (94% amino acid identity) and huPON1 (85% identity). RePON1-G3C9 has also provided the basis for the directed evolution of other recombinant variants that exhibit 10 to 380-fold higher catalytic efficiencies with various toxic OPs relative to huPON1 [13
]. The therapeutic potential of in vitro
evolved proteins, has been convincingly demonstrated with antibodies and antibody fragments [38
], but is far less developed with enzymes [39
]. Of particular concern is the toxicity of engineered proteins such as rePON1 whose sequence differs from the human protein.
In this study we examined the in vitro stability of rePON1 as a purified protein or in a complex with reconstituted HDL (rePON1-HDL), and compared them to huPON1, and huPON1-HDL (huHDL). We used various conditions that mimic physiologically relevant challenges leading to dysfunctional HDL. The results indicate significantly higher stability and reactivity of rePON1 and rePON1-HDL when compared to huPON1 and huHDL, and suggest that rePON1 and rePON1-HDL may exhibit an improved potential for in vivo treatments. To further address the issue of in vivo applicability, we generated reconstituted complexes of rePON1 with apoA-I and the phospholipids POPC (dubbed BL-3050) and evaluated the potential toxicity of BL-3050 by single administration to mice, and by repeated administrations in the course of two weeks, and observed no adverse effects. Finally, to examine whether BL-3050 is active in vivo, we applied an animal model for OP poisoning. BL-3050 and purified rePON1 were administrated few minutes, or 14 hours, prior to chlorpyriphos-oxon (CPO) poisoning to evaluate their protection abilities. Both, BL-3050 and rePON1 showed a significant protective effect in vivo. These results indicate that rePON1, and BL-3050, could be applied in vivo while taking advantage of the improved stability and catalytic efficiencies of rePON1 variants.