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Title: Mitochondrial permeability transition pore as a therapeutic target for acute pancreatitis
Author: Javed, M. A.
ISNI:       0000 0004 7657 9936
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Introduction: Acute pancreatitis (AP) is frequently complicated by pancreatic necrosis, multiple organ failure, prolonged hospitalisation and death. There is no specific, licensed drug therapy for this disease to date. Opening of the mitochondrial permeability transition pore (MPTP) is a key event in the pathophysiology of AP and is therefore a valid drug target for its treatment. Cylophilin D (CypD, a mitochondrial matrix protein), is a key regulator of MPTP formation and opening of the latter is also modulated by a new drug TRO40303. The aim of this investigation is to explore the effects of newly identified CypD inhibitors and TRO4303 in both murine and human pancreatic acinar cells (PAC) in response to established pancreatic toxins as well as the severity of experimental acute pancreatitis (EAP). The study also provides a qualitative assessment of all published literature evaluating agents tested for treatment of EAP using a newly devised scoring system. Methods: Confocal fluorescence microscopy of isolated PACs was used to evaluate the effects of MPTP inhibitors on mitochondrial function, calcium handling and cell fate in response to TLCS (taurolithocholate sulphate) and other pancreatic toxins - CCK (cholecystokinin) and POAEE (palmitoleic acid ethyl ester). In order to identify novel CypD inhibitors, computational modelling was undertaken. Compounds predicted to bind with CypD were screened using biophysical assay - surface plasmon resonance (SPR) - to evaluate binding affinity. Candidates with high affinity for CypD were subsequently evaluated in the aforementioned biological assays. Efficacy of TRO40303 in ameliorating the severity of EAP was tested in three different models. Results: TRO40303 prevented loss of mitochondrial membrane potential (ΔΨm) induced by TLCS, CCK and POAEE and improved Ca2+ clearance in PACs. TRO40303 reduced necrotic cell death pathway activation in response to TLCS in murine (p < 0.05) as well as human PACs (p < 0.01). Therapeutic administration of TRO40303 significantly reduced serum amylase, pancreatic trypsin, pancreatic and lung myeloperoxidase and histopathology scores in hyperstimulation, biliary and alcoholic EAP. Screening strategy of CypD inhibitors identified a novel small molecular inhibitor, AP-1A02, with a KD and Ki of 0.8 μM and 1.7 μM respectively. AP-1A02 protected ΔΨm and also significantly reduced necrotic cell death pathway activation in human as well as murine PACs in response to TLCS. Conclusion: This work demonstrates that MPTP is a valid target for the treatment of AP. TRO40303 protects mitochondria, reduces necrotic cell death pathway activation and ameliorates the severity of EAP and is therefore a candidate drug for human AP. Further work needs to be undertaken to optimize AP-1A02 and develop other small molecule CypD inhibitors with the aim to develop it as a potential drug for AP.
Supervisor: Sutton, Robert ; Cridddle, David ; Tepikin, Alexei Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral