Paul Gregor

Isolation, immunochemical characterization and localization of the kainate sub-class of glutamate receptor from chick cerebellum.

The low‐affinity kainate binding sites, present at high density in chick cerebellar membranes, were solubilized with Triton X‐100 and purified 41‐fold. The purified kainate binding sites, therein referred to as the kainate receptor, displayed the expected pharmacological specificity: domoate = kainate much greater than L‐glutamate much greater than D‐glutamate, quisqualate, N‐methyl‐D‐aspartate. Analysed by SDS‐PAGE under reducing conditions, a single polypeptide with a Mr = 49,000 was observed. Western blots of membranes prepared from different brain areas and animal species were analysed using a monoclonal antibody, named mAb IX‐50, raised against the purified kainate receptor. The mAb IX‐50 stained the 49,000 polypeptide in chick, goldfish and mammalian brain tissues indicating its conservation during evolution. The staining intensity correlated with the density of kainate binding sites. The mAb IX‐50 stained also a 93,000 polypeptide but the latter did not copurify with the 49,000 polypeptide. The kainate binding activity was selectively immunoadsorbed on mAb IX‐50 coupled to Sepharose which, upon elution, released a 49,000 polypeptide. The immunohistochemical localization of mAb IX‐50 binding sites in the chick cerebellar molecular layer coincided with that of the kainate receptor. We conclude that the 49,000 polypeptide is part of the kainate receptor and carries the kainate recognition site.

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties.

BACKGROUND Small molecule inhibitors of biologically important protein-glycosaminoglycan (GAG) interactions have yet to be identified. METHODS Compound libraries were screened in an assay of L-selectin-IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models. RESULTS Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity. CONCLUSIONS A new class of compounds, SMIGs, inhibits protein-GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events. GENERAL SIGNIFICANCE SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action - inhibiting protein-GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.

Kainyl-bovine serum albumin: a novel ligand of the kainate subtype of glutamate receptor with a very high binding affinity

Bovine serum albumin has been conjugated with kainylaminooxyacetylglycine to afford a multivalent kainylated protein called kainyl-bovine serum albumin (KA-BSA). This derivative, radiolabelled with 125I to more than 5000 Ci/mmol, was found to interact in the chick, goldfish and rat brain to specific membranous sites displaying the pharmacological properties attributed to the kainate sub-type of glutamate receptor. Measurements of the kinetics of association and dissociation of KA-BSA showed a quasi-irreversible binding with dissociation constants in the subpicomolar and nanomolar range. The chemical properties and the binding characteristics of KA-BSA suggest that it interacts mainly with kainate binding sites present in clusters in the membrane. Localization of the KA-BSA binding sites, by autoradiography in the chick cerebellum and by immunoperoxidase staining in the goldfish cerebellum, revealed an exclusive association with the molecular layer.

Mechanism of action and efficacy of RX-111, a thieno[2,3-c]pyridine derivative and small molecule inhibitor of protein interaction with glycosaminoglycans (SMIGs), in delayed-type hypersensitivity, TNBS-induced colitis and experimental autoimmune encephalomyelitis

Objective and designElucidate the mechanism of action of the small molecule inhibitor of protein binding to glycosaminoglycans, RX-111 and assay its anti-inflammatory activity in animal models of inflammatory disease.MaterialsThe glycosaminoglycan, heparin, was used in the mechanism of action study of RX-111. Human T lymphocytes and umbilical vein endothelial cells were used to assay the in vitro activity of RX-111. Mouse and rat models of disease were used to assay the anti-inflammatory activity of RX-111 in vivo.MethodsCircular dichroism and UV/Vis absorption spectroscopy were used to study the binding of RX-111 to the glycosaminoglycan, heparin. T lymphocyte rolling on endothelial cells under shear flow was used to assay RX-111 activity in vitro. Delayed-type hypersensitivity (DTH) and tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in mice and experimental autoimmune encephalomyelitis (EAE) in rats were used to assay anti-inflammatory activity of RX-111 in vivo.ResultsRX-111 was shown to bind directly to heparin. It inhibited leukocyte rolling on endothelial cells under shear flow and reduced inflammation in the mouse model of DTH. RX-111 was efficacious in the mouse model of inflammatory bowel disease, TNBS-induced colitis and the rat model of multiple sclerosis, EAE.ConclusionsRX-111 exercises its broad spectrum anti-inflammatory activity by a singular mechanism of action, inhibition of protein binding to the cell surface GAG, heparan sulfate. RX-111 and related thieno[2,3-c]pyridine derivatives are potential therapeutics for the treatment of inflammatory and autoimmune diseases.

RX-207, a Small Molecule Inhibitor of Protein Interaction with Glycosaminoglycans (SMIGs), Reduces Experimentally Induced Inflammation and Increases Survival Rate in Cecal Ligation and Puncture (CLP)-Induced Sepsis

The fused quinazolinone derivative, RX-207, is chemically and functionally related to small molecule inhibitors of protein binding to glycosaminoglycans (SMIGs). Composed of a planar aromatic amine scaffold, it inhibits protein binding to glycosaminoglycans (GAGs). RX-207 reduced neutrophil migration in thioglycollate-induced peritonitis (37%), inhibited carrageenan-induced paw edema (32%) and cerulein-induced pancreatitis (28%), and increased animal survival in the mouse model of cecal ligation and puncture (CLP)-induced sepsis (60%). The mechanism of RX-207 action, analyzed by UV spectroscopy, confirmed that which was elucidated for chemically related anti-inflammatory SMIGs. RX-207 binding to cell surface GAGs can account for the inhibition of neutrophil recruitment via the micro-vasculature and as a consequence, the reduction of neutrophil mediated tissue damage in the animal models of inflammation and improved survival of mice in CLP-induced sepsis.

Molecular Characterization, Ultrastructural Localization and Gene Cloning of the Chick Cerebellar Kainate Receptor

The kainate receptors mediate some of the excitarory effects of glutamate by regulating the opening of voltage independent cation selective channels. Their presence on living brain cells is nowadays monitored electrophysiologically by measuring the changes of cell membrane conductance produced by kainic and domoic acids in the absence or presence of kainate receptor antagonists such as gammaglutamyglycine and CNQX.