Deciphering Chondroitinase ABC Thermal Stability Via Molecular Dynamics

Chondroitinase ABC (chABC) is a 115 kDa bacterial lyase that has shown significant potential as a therapeutic for treating spinal cord injuries^1–5. Specifically, chABC breaks down glial scarring by degrading the side chains of chondroitin sulfate proteoglycans and has been shown to promote spinal cord plasticity, neuronal regeneration, and axonal sprouting in animal models^1,3,4. However, the impact of chABC is limited as it completely loses its activity within a few hours in dilute conditions at or above 37°C (human body temperature), thereby necessitating continuous intrathecal administration every two weeks over the course of two to six weeks to maintain therapeutic efficiency⁶. This rapid degradation poses a serious barrier for therapeutic application despite the potential of chABC. While past work performed by the Webb and Gormley labs identified incredibly promising copolymers for the stabilization of chABC, the mechanism of the stabilization is not yet clearly identified^7-9. The experimental results do not provide insight as to how the copolymers identified in the previous research endow thermodynamic stability, or how they will be impacted by other components necessary for therapy. Additionally, it must be considered that while these copolymers may provide stabilization in ideal conditions, their affects may be impacted by further chemical modification of functionalization that may be necessary for the optimization of drug-release. Therefore, it is necessary to perform further experimentation to elucidate the mechanisms of stabilization, allowing for deeper understanding and more informed implementation of future steps for therapeutic application. Molecular dynamics (MD) simulations were selected to elucidate the molecular-level interactions that lead to enhanced thermodynamic stability of chABC when in conjunction with copolymers. By conducting simulations of solely chABC in a solvated system, a baseline has been established of the temperature at which the in-silica version of this enzyme is expected to begin to degrade. At around 400K chABC in this system may begin to lose function, and at 460K and above it begins to entirely lose its original structure. This information is critical for future determinations of the stabilizing effects of the co-polymers as they are introduced to the system, as now there is a baseline of an un-stabilized system for comparison. Further work for this project currently underway has included introducing both polymers that are known to have stabilizing effects, as well as polymers that are known to not be stabilizing for similar 100 ns production MD simulations.

Citations

1. Bradbury, E. J. et al. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416, 636–640 (2002).
2. Barritt, A. W. et al. Chondroitinase ABC Promotes Sprouting of Intact and Injured Spinal Systems after Spinal Cord Injury. J. Neurosci. 26, 10856–10867 (2006).
3. Caggiano, A. O., Zimber, M. P., Ganguly, A., Blight, A. R. & Gruskin, E. A. Chondroitinase ABCI Improves Locomotion and Bladder Function following Contusion Injury of the Rat Spinal Cord. J. Neurotrauma 22, 226–239 (2005).
4. Chau, C. H. et al. Chondroitinase ABC enhances axonal regrowth through Schwann cell-seeded guidance channels after spinal cord injury. FASEB J. 18, 194–196 (2004).
5. Rosenzweig, E. S. et al. Chondroitinase improves anatomical and functional outcomes after primate spinal cord injury. Nat. Neurosci. 22, 1269–1275 (2019).
6. Tester, N. J., Plaas, A. H. & Howland, D. R. Effect of body temperature on chondroitinase ABC’s ability to cleave chondroitin sulfate glycosaminoglycans. J. Neurosci. Res. 85, 1110–1118 (2007).
7. Gormley, A. J. & Webb, M. A. Machine learning in combinatorial polymer chemistry. Nat. Rev. Mater. 6, 642–644 (2021).
8. Kosuri, S. et al. Machine-Assisted Discovery of Chondroitinase ABC Complexes toward Sustained Neural Regeneration. Adv. Healthc. Mater. 11, 2102101 (2022).
9. Tamasi, M. J. et al. Machine Learning on a Robotic Platform for the Design of Polymer–Protein Hybrids. Adv. Mater. 34, 2201809 (2022).