Enzyme-based Nerve-agent Attenuation (ATHENA)

The increasing accessibility of high-level research infrastructure capable of producing biological weapons demands improved countermeasures against exposure to these threats. Organophosphate (OP) acetylcholinesterase (AChE) inhibitors like VX and soman pose a particular risk to military personnel and can be synthesized using standard chemical processes. Developing effective delivery methods for enzyme-based therapeutics, such as phosphotriesterase (PTE), remains a high priority. However, unmodified PTE suffers from poor solution stability, susceptibility to denaturation, and a short systemic half-life. This Phase I effort will explore three stabilization strategies—directed mutagenesis, polymer-based nanoparticle encapsulation, and covalent enzyme functionalization—to generate robust PTE-based therapeutics ready for Phase II development.Directed mutagenesis of PTE has yielded highly active variants, including those developed in the Raushel lab at Texas A&M. Two variants, YT (optimized for G-type nerve agents) and L7ep-3a (enhanced catalytic activity and thermostability), were selected as starting points for stabilization efforts. These variants provide an ideal foundation for downstream modifications aimed at extending systemic circulation and improving resilience under operational conditions. Given the cost and limited availability of specialized PTE variants, early-stage stabilization work will employ horseradish peroxidase-C (HRP-C) as a model enzyme due to its similar size, isoelectric point, and lysine accessibility. Prior research at nSI demonstrated HRP-C’s strong correlation with PTE in nanoparticle loading efficiency, making it a suitable surrogate for optimizing polymer-based nanoparticle (PBNP) formulations and functionalization reactions. Polymer selection will focus on improving circulation time via PEG density tuning and the introduction of zwitterionic coatings such as carboxybetaine acrylamide (CBAA), which has shown promise in enhancing stability against repeated sarin exposure. Enzyme functionalization will further enhance stability against temperature and chemical stressors. Four approaches will be evaluated: PEGylation, conjugation to PLGA-PEG, attachment of functionalized poly(amidoamine) (PAMAM) dendrimers, and incorporation of zwitterionic polymers. These modifications aim to improve systemic circulation while maintaining enzymatic activity.nSI will leverage its expertise to generate a diverse array of stabilized PTE-based structures with enhanced resistance to thermal and chemical degradation. By employing HRP-C for rapid prototyping, we can iterate formulations efficiently without depleting scarce PTE variants. To mitigate risk, multiple experiments will be conducted per stabilization approach, ensuring robust screening of polymer size, type, and functionalization parameters. Final formulations will be transitioned to PTE variants, positioning the most promising candidates for further development in Phase II.