The goal of this proposal is to develop a sensitive chemical probe for in vivo molecular imaging of fibrin. Fibrin is a major component of thrombus (blood clot), and thrombus is implicated in a range of pathologies, e.g. ischemic stroke, myocardial infarction, pulmonary embolism, and deep vein thrombosis. Beyond hemostasis, it is known that most solid tumors are characterized by the presence of a fibrin-rich provisional matrix. Tumor invasion/metastasis absolutely requires modification of extracellular matrix components to form a suitable tumor microenvironment. Dvorak's landmark 1986 NEJM publication and subsequent research have carefully delineated the importance of the extracellular environment, and in particular, fibrin deposition, in the metastatic process. Imaging of fibrin using a fibrin-specific molecule conjugated to an optical, magnetic resonance (MR), or radioactive reporter could provide insights into the biology of all these diseases. Fibrin probes could be used to identify thrombi or tumor metastases and to guide patient management. We used a high throughput screening (HTS) assay which was implemented through the Molecular Libraries Probe Production Centers Network (MLPCN) program and resulted in the identification a lead small molecule FB-1. Initial structure-activity studies identified a compound FB-30 with 6-fold higher affinity than FB-1. Here we propose to optimize the affinity of FB-30, to derivatize ths optimized lead with an imaging reporter, and to demonstrate the utility of these chemical probes by imaging fibrin in three animal models. We will use a combination of classical medicinal chemistry and structure-based design by crystallizing the soluble fibrin degradation product DD(E). The structure-activity and structure-based data will also inform on where to conjugate an imaging reporter without adversely affecting fibrin affinity. We will incorporate a positron emittig isotope (likely F-18) into our optimized lead to create a PET imaging probe. We will also conjugate one or more gadolinium chelates into the optimized lead in order to create an MR probe. To demonstrate utility we will use imaging to evaluate these probes in animal models of thrombosis (high fibrin content) and cancer. The output of this work will be chemical probes for selective in vivo imaging of fibrin.
