Development Of Second Generation Thiol Isomerase Inhibitors To Prevent Thrombus Formation

Protein disulfide isomerase (PDI) is a widely expressed oxidoreductase that catalyzes the rearrangement of intramolecular disulfide bonds during protein maturation in the endoplasmic reticulum. However, PDI can also be secreted from both platelets and endothelial cells during thrombus formation. Inhibition of extracellular PDI in the vasculature using neutralizing antibodies or bacitracin blocks thrombus formation in pre-clinical studies. Our previous experiments identified quercetin-3-rutinoside as an inhibitor of PDI and a potent antithrombotic. Recently, we performed a high-throughput screen of the MLSMR library through the Molecular Libraries Probe Production Centers Network (MLPCN) to identify more potent and selective inhibitors of PDI. We now compare the activity of the most potent lead, ML359, to previously described PDI inhibitors and assess its antithrombotic activity.

ML359 inhibited the reductase activity of PDI in the insulin turbidometric assay with an IC₅₀ of 0.3-0.6 microM. By comparison, quercetin-3-rutinoside and juniferdin inhibited PDI with IC₅₀s of 6-10 microM, while bacitracin inhibited PDI reductase activity with an IC₅₀ of 100 microM. ML359 demonstrated no confirmed activity at <10 microM in any of the other of 380 biological assays in which it has been tested within the MLPCN. Additionally, ML359 was selective for PDI, failing to inhibit ERp5, ERp57, ERP72, thioredoxin or thioredoxin reductase activity. Inhibition of PDI by the ML359 was entirely reversible and it did not demonstrate toxicity in a HeLa cell assay.

In preparation for pre-clinical studies, further evaluation of the biophysical properties of ML359 was performed. ML359 demonstrated acceptable solubility in PBS, human plasma and GSH. However, it showed poor stability in mouse plasma as well as in presence of liver microsomes. To optimize and improve these biophysical properties of the ML359, over 85 analogs of the lead were synthesized. Modulation of the ethyl substituent of the ester group with bulky isopropyl or t-butyl substituents increased the mouse plasma stability from ∼5% in ML359 to ∼75% and ∼95% in the analogs, respectively. These substitutions only minimally altered the potency of the compound (0.6-0.9 microM). The ML359 analogs demonstrated increased inhibitory activity in platelet aggregation assays, with inhibition at 30 microM increasing from 25% for ML359 to 97 and 100% for the isopropyl and t-butyl substituted analogs, respectively. The ability of ML359 to prevent thrombus formation in a laser injury mouse model was examined. Thrombus formation was monitored following laser injury of cremaster arterioles and the effect of ML359 infusion on platelet accumulation evaluated. ML359 exposure resulted in a significantly smaller thrombus that that observed in the animals infused with vehicle alone.

ML359 and its active analogs have enhanced properties compared to the first generation PDI antagonists. These compounds are substantially more potent than previously described PDI inhibitors such as bacitracin and show significant potential as second generation inhibitors to probe PDI function in biological systems. ML359 analogs could ultimately be developed as antithrombotics that target PDI.