Introduction

1. s Comparison of the in vivo protective efficacy and thermostability of catalytic scavengers of organophosphorus nerve agents Chelsea Gaviola Mentored by Dr. Douglas M. Cerasoli and C. Linn Cadieux Results Conclusions Acknowledgments References Materials and Methods Introduction For the in vivo protective efficacy tests (Table 1), the guinea pigs pretreated with 4E9 and exposed to VX all died within minutes of exposure. The guinea pigs administered the 1.0 mg/kg 4E9 dose died within minutes of GF exposure. The survival rate against GF increased with increases in 4E9 dosage. Five out of seven guinea pigs with 10.0 mg/kg dose survived GF exposure, the highest survival rate among the 4E9 trials. All of the guinea pigs pretreated with 2D8, regardless of dosage, survived GF exposure. Seven out of seven guinea pigs with the 10.0 mg/kg 2D8 dose survived GF exposure, the highest survival rate among all trials. For the 4E9 thermostability tests (Graph 1), the TM value (the temperature where the enzyme in the cell is half folded and half unfolded; point occurs at the peak in the graph) was 72.8°C. For the 2D8 thermostability tests (Graph 2), the TM value was 71.3°C. Both enzymes continued to decrease and did not level off by the end of the DSC collection period. The purpose of this project was to assess the in vivo protective efficacy and thermostability of the PON1 mutants 4E9 and 2D8. The efficacy results in guinea pigs suggested that both 4E9 and 2D8 have protective capabilities against 2LD50s of the nerve agent GF (Table 1). The efficacy of 4E9 was enzyme dose dependent, with only the 10.0 mg/kg dose yielding the highest survival rate. In comparison, all of the tested doses of 2D8 provided protection against 2LD50s of GF. This suggested that 2D8 might be a better catalytic bioscavenger than 4E9, but many more trials need to be conducted—especially in other animal species—to confirm these conclusions. The graphs of 4E9 (Graph 1) and 2D8 (Graph 2) did not level off by the end of the DSC collection period, indicating that the enzymes were precipitating out of solution at the high temperatures. It is not possible to calculate certain thermostability and shelf-life characteristics when this occurs, but the apparent TM values suggested that both 4E9 (at 72.8°C) and 2D8 (at 71.3°C) were fairly stable. Both TM values were well above room and body temperature, the two thermal environments that the enzyme will most likely reside. In future experiments, changes in the buffer, scanning temperature, scan rates, and enzyme concentration could potentially prevent the PON1 mutants from precipitating out of solution. The shelf-life of 2D8 and 4E9 can be determined by setting the enzymes out in a storage setting and checking their stability over time. In conclusion, both 4E9 and 2D8 hold promise as potential catalytic bioscavengers because of their high protective efficacy and thermostability. From the data collected, it appears that 4E9 and 2D8 protect efficiently against GF and remain stable in a wide variety of temperatures. The project validates the research that PON1 mutants can be optimized as useful and efficient bioscavengers (Yeung et al., 2008; Yeung et al., 2004). Ultimately, the goal is to develop stable PON1 mutants that afford protection against a broad spectrum of nerve agents. If further experiments prove that 4E9 and 2D8 can provide protection against nerve agents, then these enzymes will serve as the template for new PON1 mutants. Wetherell, J., Armstrong, S., Read, R., & Clough, G. (2008). VX penetration following percutaneous poisoning: A dermal microdialysis study in the guinea pig. Toxicology Mechanisms & Methods, 18(4). Retrieved from Academic Search Premier database. Yeung, D. T., Josse, D., Nicholson, J. D., Khanal, A., McAndrew, C. W., Bahnson, B. J., Lenz, D. E., & Cerasoli, D. M. (2004). Structure/function analyses of human serum paraoxonase (HuPON1) mutants designed from a DFPase-like homology model. Biochemica et BiophysicaActa, 1702, 67-72. Yeung, D. T., Smith, J. R., Sweeney, R. E., Lenz, D. E., & Cerasoli, D. M. (2008). A gas chromatographic-mass spectrometric approach to examining stereoselective interaction of human plasma proteins with soman. Journal of Analytical Toxicology, 32, 86-91. • Dr. Douglas Cerasoli, Capstone Project Mentor • C. Linn Cadieux, Capstone Project Mentor • Dr. Tony Reeves, USAMRICD Researcher • TakwehKahjih, USAMRICD Researcher • Mrs. McDonough, Science and Mathematics Academy Faculty Advisor • Mrs. Clem, Science and Mathematics Academy Program Specialist • In vivo protective efficacy testing: • Male guinea pigs were injected with different doses (1.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 10.0 mg/kg; refer to Table 1 for exact number of guinea pigs tested per dose) of either 4E9 or 2D8 via carotid catheters • After twenty minutes, the guinea pigs were exposed subcutaneously to two median lethal doses (LD50s) of nerve agent (108.8 µg/kg of GF or 18 µg/kg of VX) • Guinea pigs were observed for up to twenty-four hours post exposure, and survival was assessed at twenty-four hours • Note: Due to safety concerns, all nerve agent handling was completed by an experienced MRICD researcher • Thermostability testing: • Sample and reference cells in the Differential Scanning Calorimeter (MicroCal VP-DSC) were rinsed thoroughly with 50 mMTris/1 mM Calcium buffer (pH 7.4) • Sample (4E9 and 2D8) and reference (50 mMTris/1 mM Calcium buffer) solutions were degassed using a ThermoVac for fifteen minutes • The reference cell was loaded with 0.5 mL of 50 mMTris/1 mM Calcium buffer using a syringe • The sample cell was loaded with 0.5 mL of either 4E9 or 2D8 using a syringe • DSC was set to scan cells from 10°C to 110°C, at a scan rate of 90°C/hour • Data were collected with the aid of the DSC computer software (VPViewer 2000) and Origin software Organophosphorus anti-cholinesterases (OPs), a subset of which are referred to as nerve agents, are highly toxic compounds that irreversibly inhibit the enzyme acetylcholinesterase (AChE) (Yeung, Smith, Sweeney, Lenz, & Cerasoli, 2008). The inhibition of AChE results in accumulation of the neurotransmitter acetylcholine (ACh), subsequently over-stimulating cholinergic receptors and inducing such symptoms as salivation, muscle spasms, twitching, and respiratory failure (Wetherell, Armstrong, Read, & Clough, 2008). Because nerve agents are extremely toxic and easy to synthesize, they have been used as chemical weapons against civilian populations and military personnel (Yeung et al., 2008). It is therefore necessary to develop effective medical countermeasures that protect against further civilian and military threats (Yeung et al., 2008). Biological scavengers have emerged as a new method to reduce the toxicity of OP nerve agents (Yeung et al., 2008). Stoichiometricbioscavengers, such as butyrylcholinesterase (BuChE), detoxify a poison by binding it in a fixed molecular ratio (Yeung et al., 2008). Catalytic bioscavengers, such as human serum paraoxonase-1 (PON1), breakdown a poison molecule, regenerate, and then repeat the process until all poison molecules have been destroyed (Yeung et al., 2008). PON1 has a low level of catalytic activity towards OPs, but with genetic alterations, it has the potential to become a useful and efficient catalytic bioscavenger (Yeung et al., 2004). Therefore, the primary purpose of this project was to assess the in vivo protective efficacy of two novel PON1 mutants, 4E9 and 2D8. It was also necessary to determine the thermostability of the mutants in order to establish what temperature ranges the enzymes could withstand. It is important for enzymes to remain folded at body and storage temperatures. Due to their specific genetic alterations, it was hypothesized that both mutants would have a higher efficacy and thermostability than wild-type PON1. Table 1: The above data table displays the results from the in vivo protective efficacy testing. The 4E9 enzyme provided no protection against 2 LD50s of VX in the guinea pigs. As the enzyme dose for 4E9 increased, the guinea pig survival rate increased, with maximum GF protection occurring at the 10.0 mg/kg dose. All of the guinea pigs injected with 2D8 survived GF exposure. 4E9 Thermostability Results 2D8 Thermostability Results Graph 1: This graph shows representative results of 4E9 thermostability analyses. The TM value occurred at the peak (72.8°C). The graph continued to decrease and did not level off before the scan ended, indicating that 4E9 was precipitating out of solution at the higher temperatures. Graph 2: This graph shows representative results of 2D8 thermostability analyses. The TM value occurred at the peak (71.3°C). The graph continued to decrease and did not level off before the scan ended, indicating that 2D8 was precipitating out of solution at the higher temperatures.