THI0019 was generated via two structural modifications to a previously identified α4β1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4β1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/β subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4β7 as well as α5β1 and αLβ2. When cross-linked to αLβ2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/β subunit interface and not at the ligand-binding site in the inserted ("I") domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.[1]

Parallel Plate Flow Detachment Assays
Recombinant human VCAM-1 (10 μg/ml or 5 μg/ml in 0.1 m NaHCO3 ) was immobilized overnight at 4 °C onto 24 × 50-mm slides cut from 15 × 100-mm polystyrene Petri dishes. The slides were washed with PBS, blocked with 2% (w/v) BSA for 2 h at room temperature, and assembled into a parallel plate flow chamber. For detachment assays, vehicle, 10 μm THI0019, 10 μg/ml mAb TS2/16, or combinations of each were mixed with Jurkat cells in low affinity running buffer, and then 2.0 × 106 cells were injected into the flow chamber and allowed to settle on the slides for 10 min. An increasing linear gradient of shear flow was pulled over the adherent cells for 300 s with the use of a computer-controlled syringe pump (Harvard Apparatus). Shear stress calculations were determined every 50 s. The shear stress in dynes/cm2 is defined as (6 μQ)/(wh2), where μ is the viscosity of the medium (0.007); Q is the flow rate in cm3/s; w is the width of the chamber (0.3175 cm), and h is the height of the chamber (0.01524 cm). The number of cells attached was recorded by digital microscopy (VI-470 charge-coupled device video camera; Optronics Engineering) at ×20 on an inverted Nikon DIAPHOT-TMD microscope every 50 s and was plotted against time.[1]

---

Cell Rolling Assays
Stromal cells (M2-10B4) were seeded on 24 × 50-mm slides cut from 125-ml tissue culture flasks, cultured overnight under standard tissue culture conditions, and assembled to a parallel plate flow chamber. TF1 cells (2.0 × 106) were mixed with vehicle or 10 μm THI0019 in running buffer and then injected into the parallel plate flow chamber system. A constant shear flow of 0.5 dynes/cm2 was applied to the system for 300 s, and the TF1 cells rolling across the stromal cell monolayer were recorded by digital microscopy. The digital recordings were then analyzed by using the Imaris Bitplane software (version 7.6.1) to determine the velocity of individual cells moving across the monolayer. The viewing area is 500 μm, and only cells that traveled at least 400 μm were included. The velocity of a cell is defined as the distance traveled divided by the time to travel that distance.[1]

---

Migration Assays
Migration assays were performed in 3-μm pore size Transwells (24 wells, Costar, Cambridge, MA). The upper chambers were pre-coated with 3 μg/ml fibronectin or 10 μg/ml VCAM-1 in 50 μl of TBS overnight at 4 °C and were then blocked with 2% BSA for 1 h at room temperature. After washing with migration medium (RPMI 1640 medium supplemented with 1% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin), the upper chambers were loaded with Jurkat cells (2 × 105 cells) in 160 μl of migration medium. The lower chambers contained 600 μl of migration medium supplemented with 10 μg/ml SDF-1α to induce chemotaxis. Jurkat cells were mixed with vehicle (1% DMSO) or THI0019 at the indicated concentrations immediately prior to being added to the upper chamber. After a 4-h incubation at 37 °C, 5% CO2, the upper chambers were removed, and cells in the lower chamber were collected and counted on a hemocytometer. Results are expressed as the total number of cells migrated.[1]

---

In Silico Docking of THI0019 in α4β7 Crystal Structure
Modeling Suite 2012 was used on a 16-core 2.4 GHz AMD Operon system to visualize THI0019 binding to an integrin model. A Glide docking model was generated starting with a crystal structure of α4β7. The structure 3v4v was downloaded from the Protein Data Bank and read into a Maestro . All chains except A (α4) and B (β7) were deleted. A basic protein preparation was performed using program defaults, with the addition of filling in missing side chains and deleting water molecules beyond 5 Å from heteroatoms. Missing atoms were identified in Ser-559 in chain A and Cys-455 in chain B. The complete preparation of the protein portion of the model involved the following: 1) basic protein preparation; 2) assignment of heteroatom states; 3) H-bond assignment, including PROPKA; 4) deletion of waters with less than three H-bonds to non-waters; 5) Impref “H-only” minimization; and 6) Impref “Minimize All” to root mean square deviation = 0.5. For THI0019, a Lig PREP calculation was performed on the structure (imported as a SDF file) by using default criteria and Epik ionization. A Glide Grid was generated using default settings based on the crystal structure ligand. In addition, a constraint was specified for metal ion. A virtual screening workflow was submitted for the crystal structure ligand and then for THI0019. The virtual screening workflow involved docking with Glide XP mode, starting with three extra conformations and rescoring with the Prime MMGBSA ΔGbind.[1]

Small molecule agonist of very late antigen-4 (VLA-4) integrin induces progenitor cell adhesion. J Biol Chem 2013 Jul 5;288(27):19414-28.

C29H35N3O7S2

601.733

601.19

C, 57.89; H, 5.86; N, 6.98; O, 18.61; S, 10.66

1378532-99-0

67704761

Solid powder

4.7

2

9

16

41

835

2

CCCC[C@@H](COC(=O)N(CC1=CC=CS1)CC2=CC=CS2)NC(=O)N[C@@H](CC(=O)OC)C3=CC4=C(C=C3)OCO4

QWDGYRDMZTZEHQ-URXFXBBRSA-N

InChI=1S/C29H35N3O7S2/c1-3-4-7-21(18-37-29(35)32(16-22-8-5-12-40-22)17-23-9-6-13-41-23)30-28(34)31-24(15-27(33)36-2)20-10-11-25-26(14-20)39-19-38-25/h5-6,8-14,21,24H,3-4,7,15-19H2,1-2H3,(H2,30,31,34)/t21-,24-/m0/s1

methyl (3S)-3-(1,3-benzodioxol-5-yl)-3-[[(2S)-1-[bis(thiophen-2-ylmethyl)carbamoyloxy]hexan-2-yl]carbamoylamino]propanoate

THI-0019; THI0019; THI 0019;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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