We currently lack therapies that reduce the risk of deep vein thrombosis without an attendant risk of bleeding or that significantly alter the development of postthrombotic syndrome. In this proposal, we postulate that P-selectin glycoprotein ligand-1 (PSGL-1), which is a ligand for P-, L-, and E-selectin provides a starting for the design of a new series of glycomimetic analogs that will suppress venous thrombosis, promote thrombus resolution, and limit late tissue remodeling events. In this regard, we have determined that GSnP-6, as a stable, high-affinity PSGL-1 mimetic, affords a unique structural scaffold for the design of a broad range of highly potent selectin-specific antagonists. Specifically, we intend to: (1) Define the potency of GSnP-6 in murine models of venous thrombosis. In the first phase of our investigations, we will define the pharmacokinetic profile of GSnP-6, as well as poly (ethylene glycol) (PEG) conjugates of GSnP-6. The dosing regimen affording the best drug delivery profile will be selected for subsequent in vivo studies. In the second phase of these investigations, the efficacy of GSnP-6 will be defined using occlusive and non-occlusive murine models of acute and chronic venous thrombosis. These studies will allow us to define the capacity of GSnP-6 to prevent the initiation and propagation of venous thrombosis, promote thrombus resolution, and limit late tissue remodeling events. (2) Design new structurally simpler PSGL-1 glycomimetics that display high selectin binding affinity. Preliminary molecular dynamics simulations have indicated that not all of the monosaccharides in the native PSGL-1 ligand or GSnP-6 contribute directly to binding affinity. This observation provides an opportunity to greatly simplify chemical synthesis by replacing non-critical residues with simpler structural analogs. MD simulations will be performed for free and bound forms of a series of proposed simplified structural variants of GSnP-6 to P-selectin. Structural variants that display the largest negative total interaction energy with P- selectin, which is predictive of a hih affinity selectin inhibitor, will be selected for synthesis, as well as characterization in vitro ad in vivo. The analog with the greatest potency will be selected for further investigation in occlusive and non-occlusive murine models of venous thrombosis. (3) Determine the effectiveness of GSnP-6 and related analogs in murine models of cancer associated venous thrombosis. Malignancy is a major risk factor for venous thromboembolism and despite optimal anticoagulation the risk of recurrent thrombosis and bleeding complications is substantially increased in this patient population. In the first phase of these investigations, the efficacy of GSnP-6 in the prevention and treatment of venous thrombosis will be assessed in tumor bearing mice. In the second phase, promising structurally simplified selectin antagonists that have been identified in Aim 2 will be assessed for their capacity to inhibit venous thrombosis and limit the development of postthrombotic syndrome in tumor bearing mice.
A screening program directed at the rational design of PSGL-1 mimetics has identified a high affinity glycomimetic, GSnP-6, that retains the target-specificity of native PSGL-1, but with far greater chemical and metabolic stability. Recent computational simulations have further enhanced our understanding of the structural biology of this glycomimetic and have confirmed its capacity to serve as a structural scaffold for the design of a broad range of highly potent selectin-specific antagonists. We believe that GSnP-6, as well as related analogs represent a promising new class of therapeutic agent for the suppression of clinically relevant, thromboinflammatory events that are responsible for the initiation and progression of deep venous thrombosis, as well as postthrombotic syndrome and chronic pulmonary hypertension.
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