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Title: Selective inhibitors of cyclophilin D to treat pancreatitis
Author: Shore, E. R.
ISNI:       0000 0004 6425 1044
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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The opening of the mitochondrial permeability transition pore (MPTP) causes cell death via necrosis in acute pancreatitis (AP) which has no curative drug treatment. Cyclophilin D (CypD) is a peptidyl prolyl cis-trans isomerase enzyme that regulates the MPTP and will be the drug target for this project. Synthesised compounds were tested using both in vitro and in vivo assays as well as employing molecular docking and high resolution X-ray crystallography to aid further compound iterations. An extensive search of the literature and re-synthesis of a selection of the identified ligands highlighted a urea based small molecule, 15, that showed micromolar activity against CypD, Ki of 5.9 µM. Compound 15 has a number of undesirable features; namely the aniline and the ethyl ester. Modification of the aniline moiety proved challenging and the potency of the starting compound 15 was lost in all cases. 37 was identified as the most promising compound with only a slight drop in potency, Ki of 10 µM. The expected benefits in metabolic stability from the flanking di-fluoro groups should outweigh this potency loss. Utilisation of the proline of the 'natural' peptide substrate for CypD to replace the ethyl ester resulted in 53b, Kd of 0.41 µM, where the pyrrolidine ring had an aryl ring with (R)- stereochemistry at the 2-position as well as an ortho-thiomethyl group. The synthesis of pyrazolidines instead of pyrrolidines resulted in the removal of the stereocentre aiding compound synthesis. The 2-aryl pyrrolidine and the 2-aryl pyrazolidine showed similar Kd values. Ultimately, the 2-aryl pyrazolidine template yielded 65b, which has an ortho-bromo substituent, and shows excellent nanomolar activity, Kd of 78 nM and Ki of 82 nM. Further optimisation of 65b gave 66d, Ki of 67 nM, which incorporates a carboxylic acid at the 5- position of 65b. Interestingly, the attempt to stabilise the 5-carboxylic acid with the incorporation of a 4-fluoro group highlighted compound 67, Ki of 92 nM, which has a benzyl ester at the 5-position rather than the carboxylic acid. Compounds 53b and 65b were capable of retaining calcium levels in freshly isolated murine mitochondria and 53b showed significant protection against the loss of ΔΨm and reduced necrosis in murine as well as human pancreatic acinar cells. We are currently awaiting further in vivo data for 65b and 67. CsA is a macrocycle that binds with excellent nanomolar potency to CypD and it has been shown that the macrocyclic structure is essential for its activity. Substitution of 15 at the R1 and the R2 positions was tolerated and this was essential for persistence with this strategy. From the preliminary studies, it was concluded that the desired stereochemistry for the CypD active site was (S, S) and the optimal ring size was the 13-membered ring system. To conclude, small molecule 15 was identified as a starting point and aniline modification identified the 2,6-difluoro compound 37 which pleasingly only resulted in a slight decrease in potency thought to be outweighed by the potential benefits in metabolic stability. Ethyl ester modification identified 65b which showed outstanding nanomolar potency; an almost 1000- fold improvement in potency when compared to the micromolar affinity of our starting molecule, 15.
Supervisor: O'Neill, P. ; Sutton, R. ; Berry, N. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral