FIGURE 5. Closing and opening α4β1 inhibitors and their conformational preferences.

(A) Overlaid size exclusion chromatograms of the α4β1 headpiece in absence (control) and presence of 10 μM drug in running buffer with Mn²⁺. (B) Drug-induced HUTS-21 LIBS epitope exposure in Jurkat T lymphoma cells in 1 mM Mg²⁺ and 1 mM Ca²⁺ (closed bars) or in 1 mM Mn²⁺ (open bars). Mean fluorescence intensity is shown as % of that with antibody 9F10 to the α4 subunit. (C-E) Competition with FITC-LDVP binding by α4β1 inhibitors and VCAM D1D2 (concentration on the y axis) to Jurkat cells in L15 medium with 1% BSA. Mean fluorescence intensity (MFI) was determined by flow cytometry without washing. (C) EO state stabilized with 4μM 9EG7 Fab and 15nM HUTS4 Fab; (D) Closed states stabilized with 5μM SG19 Fab; (E) basal ensemble. Competition experiments were done multiple times with similar results. (F) K_d and preference for the EO state $\left(K_d^c/K_d^{EO}\right)$ from C-E and for FITC-LDVP from (Li and Springer, 2018). K_d determined from competitive binding assays with nonlinear least square fits and error propagation (Eq.2, Methods). (G-I) Effect of inhibitors on the binding of conformation-specific antibodies to the β1 subunit. Binding of fluorescently labeled antibodies defined on the y axis was measured by flow cytometry without washing. EC₅₀ values are from fits to Eq.1 with errors from nonlinear least square fits (Methods). No fits are shown when the error was large (one condition each in D and I).