Identification of Roles for Peptidylarginine Deiminases (PADs) In Models of Nervous System Regeneration, Assessing Capacities of Pharmacological PAD Inhibitor Application

Human Peptidylarginine deiminases (PADs) comprise five isozymes (PAD1,2,3,4 and PAD6) performing citrullination (deimination) with distinct tissue localisation and target preferences. They are implicated in various inflammatory conditions, including those affecting the central nervous system (CNS). Although PAD2 and PAD4 are currently recognised as the main PADs associated with the CNS, less is understood about the role of PAD3, which has been noted to play roles in CNS regeneration in animal models. This thesis aimed to enhance the understanding of PADs and their role in post-translational protein citrullination during acute central nervous system (CNS) injury using human cellular models for in vitro modulation of PAD activity and citrullination in neuronal and astrocyte cell lines, respectively. The therapeutic effects of three pharmacological PAD inhibitors, the pan-PAD inhibitor Cl-amidine (with preference for PAD1, PAD3 and PAD4 inhibition), AMF30a (PAD2 inhibitor) and GSK199 (PAD4 inhibitor), were assessed under normal, hypoxic, and LPS stimulation conditions to evaluate their impact on CNS regeneration and neuroinflammation. The first objective of this research study was to establish a consistent in vitro model of CNS injury and neuronal regeneration utilising differentiated SH-SY5Y cells. PAD inhibitors were evaluated for their effects on cell migration and wound healing, using Cl-amidine, AMF30a and GSK199 critical for neuronal regeneration. The study findings suggest that PAD3 may be one of the main PAD isozymes involved in regeneration of neuronal cells, as the differentiated SH-SY5Y cells showed enhanced wound closure in response to Cl-amidine. Opposing effects were observed in the non-differentiated SHSY-5Y cells, aligning with findings for PAD inhibitor application in halting invasiveness of cancer cells and challenging the use of undifferentiated SH-SY5Y cells as a neuronal model. Astrocytic SVG-P12 cells also showed promoted wound closure with Cl-amidine, compared with PAD2 or PAD4 isozyme specific inhibitors. The second objective was to explore a controlled delivery mechanism for pan-PAD inhibitor Cl-amidine, encapsulating it into Poly-3-hydroxybutyrate microspheres which are a validated drug-delivery system with potential in CNS application. The microspheres were tested where they demonstrated biocompatibility, biodegradability and non-carcinogenicity when assessing effectiveness for the first time in a breast cancer cell line (SK-BR-3), with implications for future CNS applications. Lastly, the third objective of this research was to employ Maestro, Schrödinger Docking software, to analyze Cl-amidine’s binding affinity to PAD3 compared with available commercial inhibitors, aiming to identify FDA-approved drugs with higher affinities for PAD3, as currently, specific PAD3 inhibitors are scarce. This research paves the way for potential therapeutic interventions targeting PADs in CNS injury and other PAD-related autoimmune and inflammatory conditions.

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

PhD thesis

Grantor dc:publisher.institution

University of Westminster

Year dc:date.issued

2025

Author dc:creator

• Ahmed, D.

Advisors dc:contributor.advisor

• Lange, S.
• Getting, S.J.
• Roy
• Ashioti, M.

Identifier

oai:westminsterresearch.westminster.ac.uk:x76vx

OAI identifier oai:identifier

oai:westminsterresearch.westminster.ac.uk:x76vx