Dual-reporter high-throughput screen for small-molecule in vivo inhibitors of plasminogen activator inhibitor type-1 yields a clinical lead candidate

Abstract

Edited by John M. DenuPlasminogen activator inhibitor type-1 (PAI-1) is a serine protease inhibitor (serpin) implicated in numerous pathological processes, including coronary heart disease, arterial and venous thrombosis, and chronic fibrotic diseases. These associations have made PAI-1 an attractive pharmaceutical target. However, the complexity of the serpin inhibitory mechanism, the inherent metastability of serpins, and the high-affinity association of PAI-1 with vitronectin in vivo have made it difficult to identify pharmacologically effective small-molecule inhibitors. Moreover, the majority of current small-molecule PAI-1 inhibitors are poor pharmaceutical candidates. To this end and to find leads that can be efficiently applied to in vivo settings, we developed a dual-reporter highthroughput screen (HTS) that reduced the rate of nonspecific and promiscuous hits and identified leads that inhibit human PAI-1 in the high-protein environments present in vivo. Using this system, we screened >152,000 pure compounds and 27,000 natural product extracts (NPEs), reducing the apparent hit rate by almost 10-fold compared with previous screening approaches. Furthermore, screening in a high-protein environment permitted the identification of compounds that retained activity in both ex vivo plasma and in vivo. Following lead identification, subsequent medicinal chemistry and structure-activity relationship (SAR) studies identified a lead clinical candidate, MDI-2268, having excellent pharmacokinetics, potent activity against vitronectinbound PAI-1 in vivo, and efficacy in a murine model of venous thrombosis. This rigorous HTS approach eliminates promiscuous candidate leads, significantly accelerates the process of identifying PAI-1 inhibitors that can be rapidly deployed in vivo, and has enabled identification of a potent lead compound.

PAI-1 augments mucosal damage in colitis

“…To assess whether a small molecule inhibitor targeting PAI-1 could mimic the protection in Serpine1 -deficient mice, we used the newly developed inhibitor MDI-2268 (30), which increased the ratio of active to total tPA in vivo in both plasma and colon tissue (Fig. 5A and Fig.…”

“…5A and fig. S7A) (30). We administered this compound or a vehicle control to mice therapeutically on day 6, once mice began to lose weight from DSS-induced colitis.…”

Systemic and topical administration of spermidine accelerates skin wound healing

“…These results revealed that uPA is critical for optimal wound healing and the accelerated wound healing observed after the administration of SPD. Recently, a novel inhibitor (MDI-2268) against PAI-1, which impairs the activation of uPA-uPAR signaling, was developed and proven effective in a mouse model of deep vein thrombosis [ 38 ]. In the present study, skin wound healing in systemic SPD-treated mice was significantly promoted by the administration of MDI-2268 (Fig.…”

Systemic and topical administration of spermidine accelerates skin wound healing

“…These results revealed that uPA is critical for optimal wound healing and the accelerated wound healing observed after the administration of SPD. Recently, a novel inhibitor (MDI-2268) against PAI-1, which impairs the activation of uPA-uPAR signaling, was developed and proven effective in a mouse model of deep vein thrombosis [33]. In the present study, we investigated the effect of MDI-2268 on wound healing in SPD-treated mice.…”