Modifications around the dipeptide-mimetic core of a hydroxamic acid based matrix metalloproteinase inhibitor were studied. These variations incorporated a variety of natural, unnatural, and synthetic amino acids in addition to modifications of the P1' and P3' substituents. The results of this study indicate the following structural requirements: (1) Two key hydrogen bonds must be present between the enzyme and potent substrates. (2) Potent inhibitors must possess strong zinc-binding functionalities. (3) The potential importance of the hydrophobic group at position R3 as illustrated by its ability to impart greater relative potency against stromelysin when larger hydrophobic groups are used. (4) Requirements surrounding the nature of the amino acid appear to be more restrictive for stromelysin than for neutrophil collagenase, 72 kDa gelatinase, and 92 kDa gelatinase. These requirements may involve planar fused-ring aryl systems and possibly hydrogen-bonding capabilities.
Structural features of heparin potentially important for heparanase-inhibitory activity were examined by measuring the ability of heparin derivatives to affect the degradation of [3H]acetylated heparan sulphate by tumor cell heparanases. IC50 values were determined using an assay which distinguished degraded from undegraded substrate by precipitation of the latter with cetylpyridinium chloride (CPC). Removal of heparin's 2-O-sulphate and 3-O-sulphate groups enhanced heparanase-inhibitory activity (50%). Removal of its carboxyl groups slightly lowered the activity (18%), while combining the treatments abolished the activity. At least one negative charge on the iduronic acid/idose moiety, therefore, is necessary for heparanase-inhibitory activity. Replacing heparin's N-sulphate groups with N-acetyl groups reduced its activity (37%). Comparing this heparin derivative with 2,3-O-desulphated heparin, the placement of sulphate groups appears important for activity since the two structures have similar nominal linear charge density. In addition, unsubstituted uronic acids are nonessential for inhibition since their modification (periodate-oxidation/borohydride-reduction) enhanced rather than reduced heparanase-inhibitory activity. The most effective heparanase inhibitors (2,3-O-desulphated heparin, and [periodate-oxidized, borohydride-reduced] heparin) were tested in the chick chorioallantoic membrane (CAM) bioassay for anti-angiogenic activity and found to be at least as efficacious as heparin. 2,3-O-desulphated heparin also significantly decreased the tumor growth of a subcutaneous human pancreatic (Ca-Pan-2) adenocarcinoma in nude mice and prolonged the survival times of C57BL/6N mice in a B16-F10 melanoma experimental lung metastasis assay.
The acute inflammatory response requires that circulating leukocytes adhere to, and then migrate through, the vascular wall at the site of ijury or infection. Several receptors have been implicated in this adhesion and migration process, including the selectins, a family of carbohydrate-binding proteins. The ligand for one of these proteins, E-selectin (LECAM-2, ELAM-1) has been described by several groups to contain a polylactosamine structure bearing a terminal sialic acid residue and at least one fucose residue. We report here a more detailed investigation into the minimum structural requirements for carbohydrate recognition by E-selectin. Using both direct binding and inhibition studies we demonstrate that the sialyl Lewisx tetrasaccharides Sia(a2-3)Gal(fi1-4)[Fuc(a1-3)]GlcNAc, and Sia(a2-3)Gal(,1-4) ]Glc are the smallest oligosaccharides recognized by the lectin. In addition, an oligosaccharide containing the sialyl Lewis' epitope is also recognized, but less avidly. We propose a structural model of functional groups necessary for recognition by E-selectin, based on these data and additional experiments on modifications of sialic acid and the reducing terminal saccharide.The acute inflammatory response requires that circulating leukocytes bind to and penetrate the vascular wall to access the site of injury. Several receptors have been implicated in this interaction, including the selectins [LEC cell adhesion molecules (CAMs)], a family of carbohydrate-binding proteins. These proteins are characterized by the presence of domains with homologies to calcium-dependent lectins (Clectins), epidermal growth factor, and complement-binding proteins (1)(2)(3)(4)(5)(6). Recently, a consensus has been reached regarding the nomenclature ofthese proteins (28), resulting in the use of "selectin" for this family, and L-selectin, P-selectin, and E-selectin for the proteins LECAM-1 (LAM-1), GMP-140 (PADGEM, LECAM-3), and ELAM-1 (LECAM-2), respectively. Intense interest in these proteins has resulted in several recent publications describing aspects of the carbohydrate ligands for each of the three selectins (7,8). One member of this family, E-selectin (LECAM-2, ELAM-1) is an adhesion protein transiently expressed on the surface of vascular endothelium and has been implicated in the initial events of neutrophil extravasation. There is general agreement that the ligands for E-selectin include sialylated, polylactosamine oligosaccharides, with a fucos 'on the first {sialyl-Lewisx (sLex) epitope, Sia(a2-3)Gal(B1-4)[Fuc(a1-3)]GlcNAc-R} or second {VIM-2 epitope, Sia(a2-3)Gal(13i-4)GlcNAc(f81-3)Gal(f81-4) GlcNAc-R} N-acetylglucosamine from the nonreducing end.
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