Abstract
Plasminogen 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 high-throughput 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 vitronectin-bound 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.
Highlights
Plasminogen activator inhibitor type-1 (PAI-1) is a serine protease inhibitor implicated in numerous pathological processes, including coronary heart disease, arterial and venous thrombosis, and chronic fibrotic diseases
To address these two issues in screening for inactivators of serpins and PAI-1 in particular, we developed a dual-spectroscopic reporter screening system designed to markedly reduce the rate of compounds that falsely test positive through spectroscopic interference by mimicking the reporter substrates and to enrich for hits that retain activity in a high protein background by eliminating compounds with promiscuous binding
We found that the inhibitory activity of CCG7844BP against PAI-1 was unaffected either in ex vivo plasma or by the addition of vitronectin (Fig. 6C, open symbols) (IC50 ϭ 44 Ϯ 2.5 M in plasma compared with 50 Ϯ 3.4 or 55 Ϯ 7.5 M in BSA-containing buffer or BSA-containing buffer with vitronectin, respectively)
Summary
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. A recent series of acylanthranilic acid derivatives have shown efficacy in animal models of disease [23,24,25]; their activities against vitronectin-bound PAI-1 have yet to be reported Together, this limited collection of PAI-1 inhibitors indicates the need for further screens of diverse small-molecule libraries to identify novel inactivators of PAI-1 for in vivo applications and for the potential development of novel therapeutic candidates. We report the development of the dual-reporter system, the execution of this system in an HTS against PAI-1, and the evaluation of two novel PAI-1 inactivators in vitro and in vivo
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