A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor.

Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 10 ( 13) Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 µM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC 50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.

Synthesis and biological evaluation of quinoline salicylic acids as P-selectin antagonists.

Leukocyte recruitment of sites of inflammation and tissue injury involves leukocyte rolling along the endothelial wall, followed by firm adherence of the leukocyte, and finally transmigration of the leukocyte across cell junctions into the underlying tissue. The initial rolling step is mediated by the interaction of leukocyte glycoproteins containing active moieties such as sialyl Lewisx (sLex) with P-selectin expressed on endothelial cells. Consequently, inhibition of this interaction by means of a small molecule P-selectin antagonist is an attractive strategy for the treatment of inflammatory diseases such as arthritis. High-throughput screening of the Wyeth chemical library identified the quinoline salicylic acid class of compounds (1) as antagonists of P-selectin, with potency in in vitro and cell-based assays far superior to that of sLex. Through iterative medicinal chemistry, we identified analogues with improved P-selectin activity, decreased inhibition of dihydrooratate dehydrogenase, and acceptable CYP profiles. Lead compound 36 was efficacious in the rat AIA model of rheumatoid arthritis.

2-(4-Chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[H]quinoline-4-carboxylic acid (PSI-697): identification of a clinical candidate from the quinoline salicylic acid series of P-selectin antagonists.

P-selectin-PSGL-1 interaction causes rolling of leukocytes on the endothelial cell surface, which subsequently leads to firm adherence and leukocyte transmigration through the vessel wall into the surrounding tissues. P-selectin is upregulated on the surface of both platelets and endothelium in a variety of atherosclerosis-associated conditions. Consequently, inhibition of this interaction by means of a small molecule P-selectin antagonist is an attractive strategy for the treatment of atherosclerosis. High-throughput screening and subsequent analoging had led to the identification of compound 1 as the lead candidate. Herein, we report the continuation of this work and the discovery of a second-generation series, the tetrahydrobenzoquinoline salicylic acids. These compounds have improved pharmacokinetic properties, and a number of them have shown oral efficacy in mouse and rat models of atherogenesis and vascular injury. The lead 31 (PSI-697), is currently in clinical development for the treatment of atherothrombotic vascular events.

Quinic acid derivatives as sialyl Lewis(x)-mimicking selectin inhibitors: design, synthesis, and crystal structure in complex with E-selectin.

A search for noncarbohydrate sLe(x) mimics led to the development of quinic acid derivatives as selectin inhibitors. At Wyeth we solved the first cocrystal structure of a small molecule, quinic acid, with E-selectin. In the cocomplex two hydroxyls of quinic acid mimic the calcium-bound fucose of the tetrasaccharide sLe(x). The X-ray structure, together with structure based computational methods, was used to design quinic acid based libraries that were synthesized and evaluated for their ability to block the interaction of sLex with P-selectin. A large number of analogues were prepared using solution-phase parallel synthesis. Selected compounds showed decrease in leukocyte rolling in the IVM mouse model. Compound 2 inhibited neutrophil influx in the murine TIP model and demonstrated good plasma exposure.

Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of high-avidity beta2-integrin on neutrophils.

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewis(x) ligands and activation of beta(2)-integrins that support neutrophil arrest by binding ICAM-1. We have previously reported that neutrophils rolling on E-selectin are sufficient for signaling cell arrest through beta(2)-integrin binding of ICAM-1 in a process dependent upon ligation of L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1). Unresolved are the spatial and temporal events that occur as E-selectin binds to human neutrophils and dynamically signals the transition from neutrophil rolling to arrest. Here we show that binding of E-selectin to sialyl Lewis(x) on L-selectin and PSGL-1 drives their colocalization into membrane caps at the trailing edge of neutrophils rolling on HUVECs and on an L-cell monolayer coexpressing E-selectin and ICAM-1. Likewise, binding of recombinant E-selectin to PMNs in suspension also elicited coclustering of L-selectin and PSGL-1 that was signaled via mitogen-activated protein kinase. Binding of recombinant E-selectin signaled activation of beta(2)-integrin to high-avidity clusters and elicited efficient neutrophil capture of beta(2)-integrin ligands in shear flow. Inhibition of p38 and p42/44 mitogen-activated protein kinase blocked the cocapping of L-selectin and PSGL-1 and the subsequent clustering of high-affinity beta(2)-integrin. Taken together, the data suggest that E-selectin is unique among selectins in its capacity for clustering sialylated ligands and transducing signals leading to neutrophil arrest in shear flow.

Interaction of P-selectin and PSGL-1 generates microparticles that correct hemostasis in a mouse model of hemophilia A.

High plasma levels of soluble P-selectin are associated with thrombotic disorders and may predict future cardiovascular events. Mice with high levels of soluble P-selectin have more microparticles in their plasma than do normal mice. Here we show that chimeras of P-selectin and immunoglobulin (P-sel-Ig) induced formation of procoagulant microparticles in human blood through P-selectin glycoprotein ligand-1 (PSGL-1; encoded by the Psgl1 gene, officially known as Selpl). In addition, Psgl1-/- mice produced fewer microparticles after P-sel-Ig infusion and did not spontaneously increase their microparticle count in old age as do wild-type mice. Injected microparticles specifically bound to thrombi and thus could be involved in thrombin generation at sites of injury. Infusion of P-sel-Ig into hemophilia A mice produced a 20-fold increase over control immunoglobulin in microparticles containing tissue factor. This significantly improved the kinetics of fibrin formation in the hemophilia A mice and normalized their tail-bleeding time. P-sel-Ig treatment could become a new approach to sustained control of bleeding in hemophilia.

Rolling adhesion kinematics of yeast engineered to express selectins.

Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin-expressing yeast on a ligand-coated surface. E-selectin-expressing yeast roll specifically on surfaces bearing sialyl-Lewis-x ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large-budded yeast "walk" across the surface, alternately pivoting about bud and parent. Small-budded yeast "wobble" across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin-expressing cells are rolling rather than slipping across ligand-coated surfaces.

The N-terminal peptide of PSGL-1 can mediate adhesion to trauma-activated endothelium via P-selectin in vivo.

P-selectin glycoprotein ligand-1 (PSGL-1) is present on leukocytes and is the major ligand for endothelial expressed P-selectin. A variety of studies strongly suggests that the N-terminal region of PSGL-1 contains the binding site for P-selectin. We hypothesized that this relatively small N-terminal peptide of PSGL-1 is sufficient to support adhesion to P-selectin in vivo. To test this hypothesis, we coated 2 microm-diameter microspheres with a recombinant PSGL-1 construct, termed 19.ek.Fc. The 19.ek.Fc construct consists of the first 19 N-terminal amino acids of mature PSGL-1 linked to an enterokinase cleavage site that, in turn, is linked to human immunoglobulin G Fc. The 19.ek.Fc-coated microspheres were injected into the jugular vein of mice. Intravital microscopy of postcapillary venules within the cremaster muscle of mice revealed that a significantly greater number of 19.ek.Fc microspheres rolled compared with control microspheres. The number of rolling 19.ek.Fc microspheres was significantly diminished by pretreatment of the mice with a monoclonal antibody to P-selectin or by pretreatment of the 19.ek.Fc microspheres with a monoclonal antibody to PSGL-1. Combined, the results indicate that the N-terminal peptide of PSGL-1 can mediate adhesion to trauma-activated microvascular endothelium via P-selectin in vivo.

Beta-C-mannosides as selectin inhibitors.

Potential E- and P-selectin inhibitors were synthesized to explore a hydrophobic area on the E-selectin surface and the PSGL-1 protein binding site on the P-selectin surface that was recently defined by crystallography. Three series of mannose-based compounds (libraries A, B, and C) were synthesized using solution phase parallel synthesis. Biological evaluation of these compounds was done using two ELISA-based assays and transferred NOE (trNOE) experiments. Some of the compounds showed better activity than sLe(x) in the P-selectin assay.

L-selectin dimerization enhances tether formation to properly spaced ligand.

Selectin counterreceptors are glycoprotein scaffolds bearing multiple carbohydrate ligands with exceptional ability to tether flowing cells under disruptive shear forces. Bond clusters may facilitate formation and stabilization of selectin tethers. L-selectin ligation has been shown to enhance L-selectin rolling on endothelial surfaces. We now report that monoclonal antibodies-induced L-selectin dimerization enhances L-selectin leukocyte tethering to purified physiological L-selectin ligands and glycopeptides. Microkinetic analysis of individual tethers suggests that leukocyte rolling is enhanced through the dimerization-induced increase in tether formation, rather than by tether stabilization. Notably, L-selectin dimerization failed to augment L-selectin-mediated adhesion below a threshold ligand density, suggesting that L-selectin dimerization enhanced adhesiveness only to properly clustered ligand. In contrast, an epidermal growth factor domain substitution of L-selectin enhanced tether formation to L-selectin ligands irrespective of ligand density, suggesting that this domain controls intrinsic ligand binding properties of L-selectin without inducing L-selectin dimerization. Strikingly, at low ligand densities, where L-selectin tethering was not responsive to dimerization, elevated shear stress restored sensitivity of tethering to selectin dimerization. This is the first indication that shear stress augments effective selectin ligand density at local contact sites by promoting L-selectin encounter of immobilized ligand.