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Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the “valley of death” by bridging basic to clinical sciences.

This work examines the affinity of alpha(4)beta(1)-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between alpha(4)-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (Chen, L. L., Whitty, A., Lobb, R. R., Adams, S. P., and Pepinsky, R. B. (1999) J. Biol. Chem. 274, 13167-13175) to several cell lines and leukocytes with alpha(4)-integrin ranging from about 2,000 to 100,000 sites/cell. The results support the idea that alpha(4)-integrins exhibit multiple affinities and that affinity changes are regulated by the dissociation rate and conformation. The affinity varies by 3 orders of magnitude with the affinity induced by binding mAb TS2/16 plus Mn(2+) > Mn(2+) ' TS2/16 > activation because of occupancy of GPCR or chemokines receptor > resting receptors. A significant fraction of the receptors respond to the activating process. The change in alpha(4)-integrin affinity and the corresponding change in off rates mediated by GPCR receptor activation are rapid and transient, and their duration depends on GPCR desensitization. The affinity changes mediated by IgE receptor or interleukin-5 receptor persist longer. It appears that the physiologically active state of the alpha(4)-integrin, determined by inside-out signaling, has similar affinity in several cell types.

The leukocyte adhesion receptors, p150,95, Mac‐1 and LFA‐1 are integral membrane glycoproteins which contain distinct alpha subunits of 180,000‐150,000 Mr associated with identical beta subunits of 95,000 Mr in alpha beta complexes. p150,95 alpha subunit tryptic peptides were used to specify oligonucleotide probes and a cDNA clone of 4.7 kb containing the entire coding sequence was isolated from a size‐selected myeloid cell cDNA library. The 4.7‐kb cDNA clone encodes a signal sequence, an extracellular domain of 1081 amino acids containing 10 potential glycosylation sites, a transmembrane domain of 26 amino acids, and a C‐terminal cytoplasmic tail of 29 residues. The extracellular domain contains three tandem homologous repeats of approximately 60 amino acids with putative divalent cation‐binding sites, and four weaker repeats which lack such binding sites. The cDNA clone hybridizes with a mRNA of 4.7 kb which is induced during in vitro differentiation of myeloid cell lines. The p150,95 alpha subunit is homologous to the alpha subunits of receptors which recognize the RGD sequence in extracellular matrix components, as has previously been shown for the beta subunits, supporting the concept that receptors involved in both cell‐cell and cell‐matrix interactions belong to a single gene superfamily termed the integrins. Distinctive features of the p150,95 alpha subunit include an insertion of 126 residues N‐terminal to the putative metal binding region and a deletion of the region in which the matrix receptors are proteolytically cleaved during processing.

Integrin ␣ 4 ␤ 1 is a receptor for vascular cell adhesion molecule-1 and fibronectin. It is important in lymphopoiesis, inflammatory recruitment of leukocytes, and other situations that require cell adhesion to the vascular endothelium. The avidity of the cells expressing ␣ 4 ␤ 1 integrin can be rapidly changed by chemokines and chemoattractants. Different mechanisms, including changes in the number of interacting molecules due to the alteration of the receptor topology or changes in the affinity of the individual bonds, have been proposed to explain the nature of these fast changes in avidity. Recently, we described a fluorescent LDV-containing small molecule, which we used to monitor the affinity changes on live cells in real time (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S. et al. (2001) J. Biol. Chem. 276, 48670 -48678). Here we show that the affinity of the small molecule probe as well as the native ligand vascular cell adhesion molecule-1 varies in parallel when the integrin is modulated with divalent cations and that the affinity modulation leads to the changes in cell avidity. Using formyl peptide receptor-transfected U937 cells, we further show that the time course of avidity changes in response to the receptor activation coincides with the time course of the affinity changes. Taken together, these data are consistent with the idea that affinity regulation is a major factor that governs the avidity of cell adhesion mediated by the ␣ 4 integrin.Cell adhesion to the vascular endothelium is important for inflammation, hemostasis, cancer cell metastasis, and hostparasite interaction. Integrins and their counterstructures, the cell adhesion molecules (VCAM-1 and ICAMs), 1 together with selectins and their counterstructures (cellular mucins) determine the cell adhesive properties in these processes (1). The adhesion between endothelial cells and leukocytes is regulated by changes in the number of interacting adhesion molecules due to the difference in the expression level, molecular trafficking, and/or internalization; changes in topographical distribution due to clustering, dimerization, and other forms of molecular assembly; and changes in affinity of the individual molecules to their counterstructures (2-7). ␣ 4 ␤ 1 integrin (very late antigen-4 (VLA-4), CD49d/CD29) is one of the integrins that can mediate initial capture, rolling, and firm cell attachment to the endothelial cells (8, 9). VLA-4 is expressed on several classes of blood cells. It mediates binding to the CS-1 domain of fibronectin and to the vascular cell adhesion molecule 1 (VCAM-1), an immunoglobulin superfamily member induced by cytokines on endothelium (10, 11). ␣ 4 ␤ 1 -Integrin adhesive properties can be modulated by cytokines and chemokines, but the mechanism controlling the regulation of integrin avidity is poorly understood. In particular, it has been suggested that the changes in VLA-4-dependent adhesion are due to either th...

Abstract. The leukocyte function-associated molecule 1 (LFA-1, CDlla/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an ot subunit (Mr = 180,000) and a ~ subunit (Mr = 95,000). We report the molecular biology and protein sequence of the a subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 oL subunits and 28% identity with other integrin c~ subunits. An insertion of ~ 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/ interactive" or I domain is also present in the p150,95 and Mac-1 o~ subunits but is absent from other integrin o~ subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.T HE leukocyte function-associated 1 (LFA-1)' molecule is a member of a family of three leukocyte glycoproteins involved in cell-cell adhesion. This family of proteins, LFA-1, Mac-l, and p150,95, are heterodimers consisting of distinct ct subunits (Mr = 180,000, 170,000, and 150,000, respectively) and a common fl subunit (Mr = 95,000) (25, 47). NH2-terminal sequencing of the c~ subunits in the mouse and human has suggested that they are structurally related (33, 48); however, the cell surface expression and function of these molecules differs. LFA-1 is expressed on virtually all leukocytes and is involved in a large number of adhesion-dependent phenomena, mAb directed against LFA-1 inhibit antigen-specific T helper cell function and cytolytic functions such as cytotoxic T lymphocyte-mediated killing, antibody-dependent cytoxicity by granulocytes, and natural killer activity (47). LFA-1 is also involved 1. Abbreviations used in this paper: CME cartilage matrix protein; ECM, extracellular matrix; FNR, fibronectin receptor; ICAM-1, intracellular adhesion molecule-l; LFA, leukocyte function-associated 1; vWE von Willebrand factor. in antigen-independent interactions that mediate cell localization to sites of inflammation such as leukocyte adhesion to endothelial cells, fibroblasts, epidermal keratinocytes, and synovial cells (12-14, 17, 30, 53). Mac-1 and p150,95 are expressed on monocytes, granulocytes, and some activ...

This paper describes the use of glass and mesoporous silica microspheres (typically 1-50 µm) as supports for biomimetic lipid bilayer membrane architectures for use in biotechnological applications. We present methods and characterization of lipid bilayer membranes supported on commercially available glass beads and mesoporous silica beads formed by an aerosol process that takes advantage of self-assembly of surfactant template phases in sol-gel synthesis. Methods for controlling the concentration of fluorescent lipids, ligands, receptors, and transmembrane proteins in the bead-supported bilayer assemblies are discussed, along with methods for measuring the concentration of these species using flow cytometry. Diffusion of molecular species both within the lipid bilayer and within the mesoporous bead structure is probed using fluorescence recovery after photobleaching. Flow cytometry and confocal fluorescence microscopy are used to examine dye uptake of the porous beads and the stability of the encapsulating lipid bilayer membranes to proton and fluorophore leakage. The studies presented herein form the basis for the use of several new types of biomimetic bead-supported bilayer architectures in a variety of biotechnological applications including microimmunoassays and fluorescence-based high-throughput screening of biochemical recognition and protein function.