Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex

Abstract

The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches.

A computationally identified compound antagonizes excess FGF-23 signaling in renal tubules and a mouse model of hypophosphatemia

“…These compounds had no previously known biological functions. Thus, we have shown that our virtual high-throughput screening approach can successfully identify novel chemical probes for specific targets, complementing other recent successes using the same approach (78).…”

Computationally identified novel agonists for GPRC6A

“…The computational hits were analyzed using two criteria- i) binding energies as predicted by Vina and ii) “snapshot count” [ 43 ], giving the number of MD snapshots to which one specific compound is predicted to bind. This allowed the identification of compounds predicted to bind with high binding affinities to multiple conformations of the TM and VFT domains of GPRC6A.…”

Extended-Ensemble Docking to Probe Evolution of Ligand Binding Sites During Large-Scale Structural Changes of Proteins

“…As stated above, these structures generally represent a static snapshot of a major functional state of the target protein. More recently, the single-point docking approach has been improved to take into account thermal and conformational fluctuations of such major conformational states of the protein in an approach referred to as ensemble docking; the approach has been shown to further enhance both the quality and the efficiency of lead predictions 10 and successfully applied to a number of protein targets 11,12 (Fig. 1).…”