α4β (IC50 = 1:4 nM); α4β7 (IC50 = 2 μM); α1β1 (IC50 >100 μM); α5β1 (IC50 >100 μM); α6β1 (IC50 >100 μM); αLβ2 (IC50 >100 μM); αIIbβ3 (IC50 >100 μM)

In this study, researchers used the highly specific alpha4beta1 inhibitor 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study alpha4beta1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of alpha4beta1. Observed KD values for BIO1211 binding ranged from a value of 20-40 nM in the non-activated state of the integrin that exists in 1 mM Mg2+, 1 mM Ca2+ to 100 pM in the activated state seen in 2 mM Mn2+ to 18 pM when binding was measured after co-activation by 2 mM Mn2+ plus 10 microgram/ml of the integrin-activating monoclonal antibody TS2/16. The large range in KD values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 x 10(-4) s-1 to >140 x 10(-4) s-1. Association rate constants varied only slightly under the same conditions, all falling in the narrow range from 0.9 to 2.7 x 10(6) M-1 s-1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on alpha4beta1 that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule-1 and CS1 to alpha4beta1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.[1]

---

Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.[3]

---

Little action is seen by BIO-1211 against α1β1, α5β1, α6β1, αLβ2, and αIIbβ3 [3].

In EAE, BIO-1211 (5 and 10 mg/kg, PO, every other day) dose-independently decreases cytokine expression, leukocyte trafficking, and suppresses inflammatory responses. Significantly lower EAE clinical scores were shown by BIO-1211, which was linked to lower TNF-α, IL-17, and IFN-γ production as well as lower CD11b+ and CD45+ cell infiltration into the cerebral cortex [2].

Binding of [3H]BIO1211 to α4β1 Expressing Cells[1]
\nJurkat cells that had been enriched for α4β1 expression by FACS sorting were maintained in RPMI 1640 medium plus 10% fetal bovine serum at 37 °C in a tissue culture incubator. K562 cells that had been transfected with either the human α4, human α2, or human α1 gene and selected for high levels of α4β1, α2β1, and α1β1, respectively, by FACS were grown in the same medium supplemented with 1 mg/ml G418, 10 μg/ml gentamicin sulfate, and 50 μg/ml streptomycin. α2 and α4 K562 cells were a gift of Dr. Martin Hemler. For binding studies, the cells were pelleted by centrifugation, washed two times with TBS (50 mm Tris HCl, 150 mm NaCl, 0.1% bovine serum albumin, 2 mmglucose, 10 mm HEPES, pH 7.4), suspended at approximately 2 × 106 cells/ml in TBS, and counted using a Neubauer hemocytometer. The cells were further diluted with TBS to the concentration indicated and treated with [3H]BIO1211 at room temperature. The cells were then pelleted by centrifugation, resuspended in 100 μl of TBS plus Mn2+, and transferred to a scintillation vial containing 2.9 ml of ScintiVerse II (Fisher). Cell-associated radioactivity was quantified by scintillation counting. All studies were performed in siliconized 1.5-ml Eppendorf tubes with a standard 1-ml sample volume. Each condition was tested in at least two independent studies. In the studies indicated, a 100–1000-fold excess of unlabeled BIO1211 was added to samples after the incubation with [3H]BIO1211 to prevent further binding. Binding studies testing the effects of cell number, incubation time, and [3H]BIO1211 concentration were performed in TBS plus 2 mm MnCl2 as described. Nonspecific binding of [3H]BIO1211 to cells was assessed at each cell density and [3H]BIO1211 concentration in TBS but in the absence of added metal ion. Specific counts bound were calculated by subtracting nonspecific counts from total counts bound. Other studies testing the effects of activation on binding were performed as indicated. In the 1 mm Ca2+, 1 mmMg2+ state where K D = 20–40 nm for binding of BIO1211 to α4β1, a high background at the higher BIO1211 concentrations (3,000 cpm at 10 nm) in the standard assay format limited the concentration of [3H]BIO1211 that could be tested to ∼10 nm; however, by diluting the specific activity of the label from 50 to 5 Ci/mmol, binding could be evaluated at BIO1211 concentrations up to 100 nm, albeit with reduced precision.[1]
\nFor kinetic on rate measurements, Jurkat cells were treated with 2 nm [3H]BIO1211 at room temperature for the times indicated and then treated with a 500-fold excess of unlabeled BIO1211 to quench further binding by the [3H]BIO1211. Cells were collected by centrifugation and subjected to scintillation counting. For kinetic off rate measurements, Jurkat cells were treated with 5 nm [3H]BIO1211 at room temperature for 1 h. A 500-fold excess of unlabeled BIO1211 was added, and the cells were further incubated for the times indicated. Cells were pelleted at each time point, and cell-associated [3H]BIO1211 was measured by scintillation counting. Binding and dissociation data are represented as a percent of the maximum specific counts bound as a function of time. The data were fitted to an exponential curve by nonlinear regression. Fork off, the exponential rate constant is the off rate. For k on, the observed rate constant is the true rate constant multiplied by the [3H]BIO1211 concentration. The effect of added unlabeled BIO1211 on dissociation rates was tested over a wide range of concentrations from 100 nm (20-fold excess) to 50 μm (10,000-fold excess). The dissociation curves were superimposable over this range of concentrations, indicating that the excess unlabeled ligand was exerting no allosteric effect on the rate of dissociation (data not shown).

---

\n\nAssessing α4β1-Ligand Interactions by Competition[1]
\n[3H]BIO1211 was also used to study α4β1 function by competition, using the radioactivity as a reporter for α4β1occupancy. In this format, Jurkat cells (1 × 106/ml) in the buffers indicated were treated with serial dilutions of test compound for 1 h, and then 5 nm[3H]BIO1211, an amount sufficient to bind all unoccupied receptors, was added for 10 min before measuring the bound counts. The cells were then pelleted by centrifugation and subjected to scintillation counting. Counts bound under these conditions measure integrin that is not occupied by the test compound and is therefore free to bind the [3H]BIO1211. The competition format was also used for kinetic binding studies. Binding and dissociation constants were calculated from α4β1 that after treatment with test compound was free to bind the [3H]BIO1211.

Neuro-inflammatory responses were analyzed using qRT-PCR, western blot, and ELISA methods. Pervade of immune cells to brain was examined by Evans blue staining and immunohistochemistry (IHC) analysis of specific markers of microglia/monocytes (CD11b) and leukocytes (CD45). Decreased expression of TNF-α, IL-17, IFN-γ and pervade of CD11b+ and CD45+ cells into the cerebral cortex were observed.[2]

Animal/Disease Models: Naive C57BL/6 mice (male, 8 weeks old, weighing 20-25 grams) [2].
Doses: 5 and 10 mg/kg.
Route of Administration: Take orally every other day from the day before immunization to the 21st day after immunization.
Experimental Results: The induction of EAE can be prevented. Dramatically delayed the onset of EAE and diminished the severity of clinical EAE compared with the vehicle group. The expression of CD11b and CD45 was Dramatically diminished compared with the vehicle group. The mRNA and soluble forms of a subset of target inflammatory cytokines (IFNγ, IL-17, and TNF-α) were Dramatically diminished.

---

EAE was induced by subcutaneous immunization of myelin oligodendrocyte glycoprotein (MOG35-55) in 8-week-old C57BL/6 mice. During EAE induction, mice were separated to distinct groups and provided either BIO-1211 (5 and 10 mg/kg) or NTZ (5 mg/kg) and co-administration of these two compounds. After 21 days, neuro-inflammatory responses were analyzed using qRT-PCR, western blot, and ELISA methods. Pervade of immune cells to brain was examined by Evans blue staining and immunohistochemistry (IHC) analysis of specific markers of microglia/monocytes (CD11b) and leukocytes (CD45).[2]

[1]. Multiple activation states of integrin alpha4beta1 detected through their different affinities for a small molecule ligand. J Biol Chem. 1999 May 7;274(19):13167-75.

[2]. Prophylactic Effect of BIO-1211 Small-Molecule Antagonist of VLA-4 in the EAE Mouse Model of Multiple Sclerosis. Immunol Invest. 2015;44(7):694-712.

[3]. Selective, tight-binding inhibitors of integrin alpha4beta1 that inhibit allergic airway responses. J Med Chem. 1999 Mar 11;42(5):920-34.

Background and Objectives: The functional limitations and economic burden of therapeutic antibodies suggest that introducing alternative therapeutic compounds with the same or different mechanisms of action may be valuable. Small molecule antagonists may hold broad promise in this regard; therefore, this study aimed to investigate the preventive effect of BIO-1211 (a very late antigen-4 (VLA4) blocker) in a mouse model of experimental autoimmune encephalomyelitis (EAE) caused by multiple sclerosis and to compare it with the commercially available drug natezumab (NTZ). Methods: EAE was induced in 8-week-old C57BL/6 mice by subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG35-55). During EAE induction, mice were divided into different groups and administered BIO-1211 (5 and 10 mg/kg) or NTZ (5 mg/kg), or a combination of both compounds. After 21 days, neuroinflammatory responses were analyzed using qRT-PCR, Western blot, and ELISA. We assessed the infiltration of immune cells into the brain by detecting specific markers of microglia/monocytes (CD11b) and leukocytes (CD45) using Evans blue staining and immunohistochemical (IHC) analysis. Results: In mice, targeting and disrupting the VLA4/VCAM1 interaction with the BIO-1211 agonist reduced cytokine expression, leukocyte migration, and suppressed the inflammatory response in an EAE model (p < 0.01), and this effect was dose-independent (data not shown). Mice treated with a combination of BIO-1211 and NTZ showed significantly lower EAE clinical scores, which were associated with decreased expression of TNF-α, IL-17, and IFN-γ, and reduced infiltration of CD11b(+) and CD45(+) cells into the cerebral cortex. Conclusion: Our results suggest that the BIO12-11 compound can serve as an effective tool for further understanding the biological function of VLA4/VCAM1 interaction and can be considered an EAE inhibitor. [2]

---

Although many detection methods have been used to study the function of α4β1, the data generated by [3H]BIO1211 revealed a variety of features of ligand binding that traditional detection methods could not reveal. The most important observation is that activation is not defined by a single state, but by several different states that produce a range of affinities, and the differences in affinity are closely related to the dissociation rate of the integrin-ligand complex. As a soluble monovalent probe of α4β1 function, BIO1211 and its associated inhibitors represent new tools that can help to further reveal the complexities associated with integrin activation. [1]

C36H48N6O9

708.80112

708.348

C, 61.00; H, 6.83; N, 11.86; O, 20.31

187735-94-0

9961766

White to off-white solid powder

5.433

7

9

16

51

1250

4

CC1=CC=CC=C1NC(=O)NC2=CC=C(C=C2)CC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)N3CCC[C@H]3C(=O)O

NVVGCQABIHSJSQ-KFZSMJGVSA-N

InChI=1S/C36H48N6O9/c1-20(2)17-26(38-29(43)18-23-12-14-24(15-13-23)37-36(51)40-25-10-7-6-9-22(25)5)32(46)39-27(19-30(44)45)33(47)41-31(21(3)4)34(48)42-16-8-11-28(42)35(49)50/h6-7,9-10,12-15,20-21,26-28,31H,8,11,16-19H2,1-5H3,(H,38,43)(H,39,46)(H,41,47)(H,44,45)(H,49,50)(H2,37,40,51)/t26-,27-,28-,31-/m0/s1

(2S)-1-[(2S)-2-[[(2S)-3-carboxy-2-[[(2S)-4-methyl-2-[[2-[4-[(2-methylphenyl)carbamoylamino]phenyl]acetyl]amino]pentanoyl]amino]propanoyl]amino]-3-methylbutanoyl]pyrrolidine-2-carboxylic acid

BIO-1211; 187735-94-0; BIO 1211; UNII-61G4E2353I; 61G4E2353I; CHEMBL88478; L-Proline, N-((4-((((2-methylphenyl)amino)carbonyl)amino)phenyl)acetyl)-L-leucyl-L-alpha-aspartyl-L-valyl-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~352.71 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.93 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (2.93 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (2.93 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)