Discovery of very late antigen-4 (VLA-4, alpha4beta1 integrin) allosteric antagonists

Abstract

Integrins are cell adhesion receptors that mediate cell-to-cell, or cell-to-extracellular matrix adhesion. They represent an attractive target for treatment of multiple diseases. Two classes of small molecule integrin inhibitors have been developed. Competitive antagonists bind directly to the integrin ligand binding pocket and thus disrupt the ligand-receptor interaction. Allosteric antagonists have been developed primarily for α(L)β(2)- integrin (LFA-1, lymphocyte function-associated antigen-1). Here we present the results of screening the Prestwick Chemical Library using a recently developed assay for the detection of α(4)β(1)-integrin allosteric antagonists. Secondary assays confirmed that the compounds identified: 1) do not behave like competitive (direct) antagonists; 2) decrease ligand binding affinity for VLA-4 ∼2 orders of magnitude; 3) exhibit antagonistic properties at low temperature. In a cell based adhesion assay in vitro, the compounds rapidly disrupted cellular aggregates. In accord with reports that VLA-4 antagonists in vivo induce mobilization of hematopoietic progenitors into the peripheral blood, we found that administration of one of the compounds significantly increased the number of colony-forming units in mice. This effect was comparable to AMD3100, a well known progenitor mobilizing agent. Because all the identified compounds are structurally related, previously used, or currently marketed drugs, this result opens a range of therapeutic possibilities for VLA-4-related pathologies.

FIGURE 1.

Binding and dissociation of the LDV-FITC probe in response to the addition of screening hits. A, LDV-FITC probe binding and dissociation on U937 cells plotted as mean channel fluorescence (FL1) versus time. The experiment involved sequential additions of fluorescent LDV-FITC probe (25 nm), LDV (1 μm, control, excess of unlabeled competitor), or different concentrations of compounds tested. One representative experiment out of two experiments for each compound is shown. B, steady state value of the LDV-FITC fluorescence obtained in experiments analogous to the one shown in panel A, 300–400 s after compound addition, plotted versus compound concentration. LDV-FITC fluorescence was normalized assuming that the value of fluorescence after LDV addition is equal to 0, and after DMSO addition (vehicle) is equal to 1. The data represent means + S.E. (n = 2) for two independent experiments. Curves represent a fit to a sigmoidal dose response equation (variable slope) performed using GraphPad Prism software. EC₅₀ values and compound structures are shown on the right.

FIGURE 2.

Binding and dissociation of the LDV-FITC probe in response to the addition of screening hits. A, LDV-FITC probe binding and dissociation on U937 cells stably transfected with the non-desensitizing mutant of FPR plotted as mean channel fluorescence (FL1) versus time. The experiment involved sequential additions of the fluorescent LDV-FITC probe (4 nm), fMLFF (100 nm), LDV (control, excess unlabeled competitor), or saturating concentrations of compounds tested. One representative experiment (for trifluoperazine) out of two experiments for each compound is shown. B, LDV-FITC probe binding and dissociation on U937 cells plotted as mean channel fluorescence (FL1) versus time at low temperature (15 °C). The experiment involved sequential additions of the fluorescent LDV-FITC probe (25 nm), LDV (control, excess unlabeled competitor), or saturating concentration of compounds tested. One representative experiment (for trifluoperazine) of two experiments for each compound is shown. C, LDV-FITC “dissociation rates” (k_off) obtained in kinetic experiments analogous to the experiments shown in panels A and B. The dissociation components of the curves were fitted to a single exponential equation using GraphPad Prism software and plotted for different compounds. Control represents the actual dissociation rates obtained using excess unlabeled competitor (LDV). Notice that for all treatment conditions k_off values were larger than in the control sample, representing faster dissociation of the probe.

FIGURE 3.

Reversibility of compound binding assessed using binding and dissociation of the LDV-FITC probe in response to the addition of screening hits. A, LDV-FITC probe binding and dissociation on U937 cells plotted as mean channel fluorescence (FL1) versus time. The experiment involved sequential additions of fluorescent LDV-FITC probe (25 nm), and the compounds tested (30 μm). After dissociation of the LDV-FITC probe the cells were washed three times in RPMI media to remove all traces of the compound. Next, the LDV-FITC probe was replenished, followed by addition of the competitor (LDV). Notice that after the wash step, the binding of LDV-FITC was identical to the binding after the first addition. This indicates that the compound blocking LDV-FITC binding after the first addition was completely removed by the wash. Thus, the binding of the compound was reversible. Analogous data were obtained for all five compounds tested (see Fig. 1 for the list). B, the same experiment as described in panel A was performed using forskolin, an activator of adenylyl cyclase. The addition of forskolin was insufficient to induce the dissociation of LDV-FITC probe to the baseline (indicated by dashed line). A small decrease in the probe binding is attributed to a small number of constitutively active VLA-4 present on the cell surface (a fraction of VLA-4 with slow LDV-FITC dissociation, see panel A after LDV addition). Forskolin treatment reduced the binding affinity for these active receptors to the resting state. Note the slower effect after forskolin addition, and the irreversible inhibition of LDV-FITC that involve intracellular signaling.

FIGURE 4.

Kinetics of real-time binding of HUTS-21 (PE) antibodies to U937 cells. Real-time binding of HUTS-21 (LIBS) antibodies plotted as mean channel fluorescence (FL2) versus time. The addition of HUTS-21 antibodies (first arrow) resulted in rapid nonspecific binding of antibodies. The addition of increasing amounts of LDV ligand (arrows) resulted in increased rates of antibody binding in the absence (red), or in the presence of the compound (blue). Compound was added at 0 time point. Notice that binding of HUTS-21 in the absence of the compound starts at 1 nm LDV (red arrowhead). To induce similar binding of HUTS-21 in the presence of the compound 0.1 μm of LDV was required (blue arrowhead). One representative experiment (for perphenazine) is shown. Analogous data were obtained for all five compounds tested (see Fig. 1 for the list).

FIGURE 5.

Changes in cell adhesion between VLA-4-expressing U937 cells and VCAM-1-transfected B78H1 cells in response to compound addition. U937 cells were stained using a red fluorescent dye (PKH26), and B78H1/VCAM-1 cells were stained using a green fluorescent dye (PKH67). Cells were mixed at 37 °C and sampled continuously using a flow cytometer. Double positive (red and green) cell aggregates were followed as described under “Experimental Procedures,” and plotted as % of aggregates (% Agg) versus time. 30 μm individual compounds (A, B, and C) or 1 μm of LDV competitor (D) were added. Notice the rapid decrease in the number of cell aggregates after the addition of allosteric antagonists. A representative experiment of two experiments is shown.