Design, synthesis and binding properties of a fluorescent α9β1/α4β1 integrin antagonist and its application as an in vivo probe for bone marrow haemopoietic stem cells

Abstract

The α₉β₁ and α₄β₁ integrin subtypes are expressed on bone marrow haemopoietic stem cells and have important roles in stem cell regulation and trafficking. Although the roles of α₄β₁ integrin have been thoroughly investigated with respect to HSC function, the role of α₉β₁ integrin remains poorly characterised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to determine cell-associated protein localisation and activation, and to elucidate the mechanism of small molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent cell binding properties of a new fluorescent α₉β₁ integrin antagonist (R-BC154), which was based on a series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154 exhibits high nanomolar binding affinities to α₉β₁ integrin with potent cross-reactivity against α₄β₁ integrin under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differences in the binding kinetics between α₉β₁ and α₄β₁ integrins, which showed faster binding to α₄β₁ integrin relative to α₉β₁, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1) screening small molecule inhibitors of α₉β₁ and α₄β₁ integrins; (2) investigating the biochemical properties of α₉β₁ and α₄β₁ integrin binding and (3) investigating integrin expression and activation on defined cell phenotypes in vivo.