Interrogating a Compound Library in Search of an Inhibitor for TREM-like Transcript-1 to Fibrinogen Binding.

Area of Science:

• Biochemistry
• Pharmacology
• Cardiovascular Research

Background:

• Cardiovascular disease (CVD) is a leading global cause of mortality, often driven by aberrant platelet function and thrombus formation.
• TREM-like transcript-1 (TLT-1), a platelet-released receptor, binds fibrinogen and von Willebrand factor, playing a critical role in thrombosis.
• TLT-1 presents a potential therapeutic target for preventing CVD-related thrombosis.

Purpose of the Study:

• To design and implement a high-throughput assay for screening small molecule inhibitors of TLT-1 and fibrinogen binding.
• To identify novel compounds that can inhibit TLT-1's pro-thrombotic activity.
• To validate the efficacy and safety of identified inhibitor compounds.

Main Methods:

• Developed an attachment assay using HEK-293 cells expressing human TLT-1 to screen 800 small molecules for inhibition of TLT-1/fibrinogen binding.
• Utilized crystal violet staining for assessing cell adhesion and inhibition.
• Employed MTT and calcein AM staining assays to evaluate compound cytotoxicity and confirm inhibition was not due to cell death.

Main Results:

• The assay demonstrated that TLT-1 expression increased cell adhesion by over 2-fold.
• Approximately 80 compounds were identified, inhibiting TLT-1/fibrinogen binding by more than 80%.
• Cytotoxicity assays confirmed that the observed inhibition was not a result of compound toxicity. Four compounds showed potential, with BM-8372 significantly affecting platelet aggregation.

Conclusions:

• A robust assay was established for screening inhibitors of TLT-1, a critical mediator of thrombosis.
• The screening identified potent small molecule inhibitors of TLT-1/fibrinogen binding, with minimal cytotoxicity.
• These findings provide promising therapeutic candidates for developing novel anti-thrombotic strategies to combat cardiovascular disease.