Thirty minutes later, the medium containing monocytes were aspired and the unattached monocytes were carefully washed out with PBS. progressive disease, and its clinical manifestations include coronary artery disease, cerebrovascular disease and peripheral arterial disease. The interaction between the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and oxidatively modified low-density lipoprotein (ox-LDL) plays a significant role in the pathobiology of atherosclerosis, as well as myocardial ischemia and hypertension1. Conditions leading to atherosclerosis, such as diabetes, hypertension and dyslipidemia, are associated with a global increase in inflammatory signaling and generation of reactive oxygen species (ROS), leading to enhancement of LDL oxidation. LOX-1 mediates the recognition and internalization of ox-LDL by vascular endothelial cells2. Of all known scavenger receptors, LOX-1 is the predominant scavenger receptor in vascular endothelial cell3,4. Recent studies show that blockade of LOX-1 by anti-sense oligo or antibody may reduce myocardial ischemic injury, genesis of hypertension and extent of atherosclerosis5,6,7,8. Hence, LOX-1 is an attractive target for the therapy of a number of cardiovascular disease states9. LOX-1 is a transmembrane protein comprising four domains and the C-terminal AST-6 domain is responsible for ox-LDL recognition10,11. The crystal structure analysis of the C-terminal domain of human LOX-1 (Fig. 1A,B) suggests that it exists as a Edem1 homodimer with a central hydrophobic tunnel that extends through the entire molecule12,13. Open in a separate window Figure 1 The structure of LOX-1 and its interaction with ox-LDL.(A) A cartoon showing the domain structure of LOX-1. LOX-1 is a transmembrane protein with 273 residues comprising 4 domains. The first 36 residues form a cytoplasmic tail, followed by a single transmembrane domain (21 residues), and an extracellular region comprising two domains. The first one (58C142) is predicted to be a coil, and the second (143C273) is a C-type lectin-like domain (CTLD) responsible for ox-LDL recognition and it exists as a disulfide-linked homodimer12,13. (B) Left panel: A view of the surface representation of the C-terminal domain exhibiting the central tunnel. Right panel: A rotated view of the surface showing basic spine, the linear arrangement of basic residues. (C) A flow chart showing steps involved in virtual screening. Under oxidative stress, LDL undergoes changes making its surface electronegative. The most significant modification pertinent to LOX-1 interaction is the covalent attachment of a phospholipid moiety on the Lys side chains of apolipoprotein B-100, a component of LDL13,14,15. Modeling studies have shown that the phospholipid moiety fits well into the hydrophobic tunnel of LOX-116. Binding measurements suggest that it ox-LDL binds to LOX-1 with high affinity. Additionally, mutations of certain residues present in the tunnel impair binding to ox-LDL, confirming the crucial role AST-6 of the tunnel in ligand recognition and binding16. Another structural feature relevant to ox-LDL binding is called basic spine, AST-6 and it is a linear arrangement of basic residues across the dimer surface (Fig. 1B)13,15. It has been proposed that these residues interact with a long helix of the apolipoprotein B protein of LDL15. Inhibition of ox-LDL binding with LOX-1 can be achieved by either blocking the basic spine or the tunnel, but a ligand can have more interactions in a tunnel than on the surface resulting in higher affinity and specificity. AST-6 Therefore we searched for molecules that can occupy the tunnel. Some investigators have described substrate mimics or natural inhibitors that inhibit LOX-1 activity, albeit in large concentrations17,18,19,20,21,22. The present paper describes identification of chemical inhibitors of LOX-1 and their efficacy in blocking LOX-1 expression and activity in nanomolar quantities. We used structure-based drug design (SBDD) techniques for finding inhibitors of LOX-1 that stop the hydrophobic tunnel and stop ox-LDL/LOX-1 connections. Towards this objective, we explored a different chemical space supplied by the ZINC data bottom without restricting ourselves to substrate analogues or organic items23. After determining several lead substances, we evaluated the experience of the very best five applicants and noticed that two of these exerted an extremely significant.