Compounds for modulating integrin CD11B/CD18
View Patent ↗The application describes an assay for the identification of small molecule modulators of integrin CD11b/CD18 and small molecules capable of modulating activity of this receptor. Such compounds may be used in certain embodiments for treating a disease or condition selected from inflammation, immune-related disorders, cancer, ischemia-reperfusion injury, stroke, neointimal thickening associated with vascular injury, bullous pemphigoid, neonatal obstructive nephropathy, and cardiovascular disease, or in other embodiments for the treatment of a disease or condition selected from immune deficiency, acquired immune deficiency syndrome (AIDS), myeloperoxidase deficiency, Wiskott-Aldrich syndrome, chronic granulomatous disease, hyper-IgM syndromes, leukocyte adhesion deficiency, Chediak-Higashi syndrome, and severe combined immunodeficiency.
1. A no-wash, cell-based assay for the identification of small molecule modulators of integrin CD11b/CD18, the assay consisting of:
contacting cells with a solution comprising a test compound on a substrate treated with a compound that affects cell adhesion;
inverting the substrate such that non-adherent cells move away from the substrate by the action of gravity;
removing the solution from the substrate after inverting the substrate without contacting the substrate with an additional liquid to wash or rinse the substrate; and
detecting adherent cells on the substrate, wherein detecting adherent cells comprises measuring the viability of the adherent cells or imaging the adherent cells.
2. The no-wash, cell-based assay of claim 1 , wherein the cells are K562 cells expressing integrin CD11b/CD18.
3. The no-wash, cell-based assay of claim 1 , wherein the compound that affects cell adhesion promotes cell adhesion to the substrate.
4. The no-wash, cell-based assay of claim 3 , wherein the compound that affects cell adhesion is selected from the group consisting of fibrinogen, iCb3, factor X, ICAM-1, NIF, and denatured BSA.
5. The no-wash, cell-based assay of claim 3 , wherein the solution further comprises Mg 2+ , Ca 2+ , or a mixture thereof.
6. The no-wash, cell-based assay of claim 1 , wherein the solution is removed from the substrate by aspirating the solution.
7. The no-wash, cell-adhesion-based assay of claim 1 , wherein the cells are treated with a fixative prior to removing the solution from the substrate.
8. The no-wash, cell-adhesion-based assay of claim 7 , wherein the fixative is formaldehyde.
9. A no-wash, cell-adhesion-based assay for the identification of small molecule modulators of integrin CD11b/CD18, the assay consisting of:
contacting cells with a solution comprising a test compound on a substrate treated with a compound that affects cell adhesion;
inverting the substrate such that non-adherent cells move away from the substrate by the action of gravity without contacting the substrate with an additional liquid to wash or rinse the substrate;
and detecting adherent cells on the substrate.
10. The no-wash, cell-adhesion-based assay of claim 9 , wherein the cells are K562 cells expressing integrin CD11b/CD18.
11. The no-wash, cell-adhesion-based assay of claim 9 , wherein the compound that affects cell adhesion promotes cell adhesion to the substrate.
12. The no-wash, cell-adhesion-based assay of claim 11 , wherein the compound that affects cell adhesion is selected from the group consisting of fibrinogen, iCb3, factor X, ICAM-1, NIF, and denatured BSA.
13. The no-wash, cell-adhesion-based assay of claim 11 , wherein the solution further comprises Mg 2+ , Ca 2+ , or a mixture thereof.
14. The no-wash, cell-adhesion-based assay of claim 9 , wherein detecting adherent cells comprises measuring the viability of the adherent cells or imaging the adherent cells.