Cytoprotective Drug-Tissue Plasminogen Activator Protease Interaction Assays: Screening of Two Novel Cytoprotective...

Tissue plasminogen activator (tPA) is currently used in combination with endovascular procedures to enhance recanalization and cerebral reperfusion and is also currently administered as standard-of-care thrombolytic therapy to patients within 3-4.5 h of an ischemic stroke. Since tPA is not neuroprotective or cytoprotective, adjuvant therapy with a neuroprotective or an optimized cytoprotective compound is required to provide the best care to stroke victims to maximally promote clinical recovery. In this article, we describe the use of a sensitive standardized protease assay with CH₃SO₂-D-hexahydrotyrosine-Gly-Arg-p-nitroanilide•AcOH, a chromogenic protease substrate that is cleaved to 4-nitroaniline (p-nitroaniline) and measured spectrophotometrically at 405 nm (OD_405 nm), and how the assay can be used as an effective screening assay to study drug-tPA interactions. While we focus on two compounds of interest in our drug development pipeline, the assay is broadly applicable to all small molecule neuroprotective or cytoprotective compounds currently being discovered and developed worldwide. In this present study, we found that the specific tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1; 0.25 μM), significantly (p < 0.0001) inhibited 4-nitroaniline release, by 97.74% during the 10-min duration of the assay, which is indicative of tPA protease inhibition. In addition, two lead chromone cytoprotective candidates, 2-(3',4',5'-trihydroxyphenyl)chromen-4-one (3',4',5'-trihydroxyflavone) (CSMC-19) and 3-hydroxy-2-[3-hydroxy-4-(pyrrolidin-1-yl)phenyl]benzo[h]chromen-4-one (CSMC-140), also significantly (p < 0.05) reduced 4-nitroaniline accumulation, but to a lesser extent. The reduction was 68 and 45%, respectively, at 10 μM, and extrapolated IC₅₀ values were 4.37 and >10 μM for CSMC-19 and CSMC-140, respectively. Using bonafide 4-nitroaniline, we then demonstrated that the reduction of 4-nitroaniline detection was not due to drug-4-nitroaniline quenching of signal detection at OD_405 nm. In conclusion, the results suggest that high concentrations of both cytoprotectives reduced 4-nitroaniline production in vitro, but the inhibition only occurs with concentrations 104-1025-fold that of EC₅₀ values in an efficacy assay. Thus, CSMC-19 and CSMC-140 should be further developed and evaluated in embolic stroke models in the absence or presence of a thrombolytic. If necessary, they could be administered once effective tPA thrombolysis has been confirmed to avoid the possibility that the chromone will reduce the efficacy of tPA in patients. Stroke investigator developing new cytoprotective small molecules should consider adding this sensitive assay to their development and screening repertoire to assess possible drug-tPA interactions in vitro as a de-risking step.