Characterization of novel inhibitors of thiol isomerases

Bekendam, R.H. (2014) Characterization of novel inhibitors of thiol isomerases. thesis, Medicine.

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Abstract

Identification of novel thiol isomerase inhibitors as potent antithrombotics Protein disulfide isomerase (PDI) is an oxidoreductase localized to the endoplasmic reticulum where it catalyzes disulfide bond rearrangements required for proper protein folding. PDI can also be secreted into the extracellular environment. Recently it has been shown that extracellular PDI is critical for thrombus formation following vascular injury. However, the mechanism by which PDI contributes to thrombosis is unknown. Next to its function as an oxidoreductase, PDI also demonstrates de- and transnitrosylation activity. PDI can function as a nitrosylase, adding or removing nitric oxide (NO) from target proteins and facilitating the transfer of NO from the extracellular environment into cytosol. Recently, a high-throughput screen of the Molecular Libraries Small Molecule Repository (MLSMR) was performed to identify more potent and selective inhibitors of PDI and ERp5 (another member of the thiol isomerase family). The most potent leads, ML-359, a bromo-indole and S775665 are here compared and assessed for antithrombotic activity. All three probes inhibited PDI in the insulin turbidometric assay with an IC50 in the low micromolar range. ML-359 and the bromo-indole showed selectivity for PDI within the thiol isomerase family. However, S775665 inhibited ERp5 and PDI with a similar IC50. ML359 and the bromo indole showed ~50% inhibition in ex vivo platelet aggregation studies. Methylation of ester moieties within the ML359 increased its plasma stability and inhibitory effect on platelet aggregation to ~97%. S775665 showed total inhibition in ex vivo platelet aggregation. Potent analogs of ML359 showed to be reversible inhibitors in platelet aggregation studies. However, the S775665 probe showed to be an irreversible inhibitor. Neither ML359 nor the S775665 probe affected the transnitrosylation activity of PDI on the cell surface of platelets. However, the bromo-indole did inhibit PDI transnitrosylation. In this report, we show that different novel inhibitors of PDI and other thiol isomerases have distinct activities when tested in a battery of functional assays. Ideal small molecules should inhibit the prothrombotic properties of PDI and preserve its antithrombotic transnitrosylation activity. ML359 demonstrates this inhibitory profile and is a superior candidate for pre-clinical testing.

Item Type:	Thesis (Thesis)
Supervisor name:	Son, W.J. van M.D. and UMCG / University of Groningen
Supervisor name:	Flaumenhaft, R. and Associate Professor and BIDMC / Harvard Medical School
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 10:54
Last Modified:	25 Jun 2020 10:54
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/1474

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