Spectrophotometric coupled-enzyme assay

A fixed concentration of enzyme (15 nM of p38α or p38β) was incubated with VX-745 in DMSO for 10 mins at 30 ℃ in 0.1 M HEPES buffer, pH 7.5, containing 10% glycerol, 10 mM MgCl2, 2.5 mM phosphoenolpyruvate, 200 μM NADH, 150 μg/mL pyruvate kinase, 50 μg/mL lactate dehydrogenase and 200 μM EGF receptor peptide (KRELVEPLTPSGEAPNQALLR). The reaction was initiated with 100 μM and 70 μM ATP for p38α and p38β assays, respectively. The decrease of absorbance at 340 nm was monitored to follow the rate of the reaction. IC50 was evaluated from the rate data as a function of the inhibitor concentration.

Cell lines

Human bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells

Preparation method

The solubility of this compound in DMSO is > 21.8 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

60 nM ~ 20 μM; 48 hrs

Applications

In BMSCs, VX-745 inhibited IL-6 and VEGF secretion, without affecting their viability. VX-745 also reduced TNF-α-induced IL-6 secretion in BMSCs. In addition, VX-745 inhibited both MM cell proliferation and IL-6 secretion in BMSCs induced by adherence of MM cells to BMSCs, which implied that VX-745 could inhibit paracrine MM cell growth in the bone marrow milieu, as well as overcome cell adhesion-related drug resistance.

Animal models

A type II collagen-induced arthritis (CIA) mice model

Dosage form

2.5, 5 and 10 mg/kg; p.o.; b.i.d., for 20 days

Applications

In a CIA mice model, VX-745 at the doses of 2.5, 5, and 10 mg/kg improved the inflammatory scores by 27%, 31% and 44%, respectively. In addition, compared with the vehicle control group, VX-745 also improved the histological scores by 32 ~ 39%, which indicated its protection on bone and cartilage erosion.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

VX-745 is a first-generation inhibitor of p38αMAPK with IC50 value of 1.0 μM when tested with Werner syndrome dermal fibroblasts [1].

Mammalian mitogen-activated protein (MAP) kinases are serine /threonine kinases and have 4 subfamilies: ERK, JNK, MAPKp38 and ERK5. MAPK pathway plays a pivotal role in transmitting signals from cell membrane to nucleus and also participates in many intracellular signaling pathways that control a wide spectrum of cellular processes (growth, differentiation, stress responses, cancer progression) [2]. MAPKp38 is originally isolated from lipopolysaccharide-stimulated monocytes. And according to the distribution in tissue, regulation of kinase activation and subsequent phosphorylation of downstream substrates, it is divided into four isoforms p38alpha, p38beta, p38gamma and p38delta [3].

VX-745 is an exquisite kinase selectively inhibit p38αMAPK and is regarded as an anti-inflammatory candidate. When tested with PBMCs or whole blood, VX-745 treatment could inhibit the secretion of IL-1β and TNF-α in vitro [4]. In human dermal fibroblast cells, VX-745 treatment blocked p38 signaling to rescue the aging phenotype in Werner syndrome [5].

References:
[1].  Bagley, M.C., et al., Microwave-assisted Ullmann C-S bond formation: synthesis of the P38alpha MAPK clinical candidate VX-745. J Org Chem, 2009. 74(21): p. 8336-42.
[2].  Dent, P., et al., MAPK pathways in radiation responses. Oncogene, 2003. 22(37): p. 5885-96.
[3].  Dominguez, C., D.A. Powers, and N. Tamayo, p38 MAP kinase inhibitors: many are made, but few are chosen. Curr Opin Drug Discov Devel, 2005. 8(4): p. 421-30.
[4].  Duffy, J.P., et al., The Discovery of VX-745: A Novel and Selective p38alpha Kinase Inhibitor. ACS Med Chem Lett, 2011. 2(10): p. 758-63.
[5].  Bagley, M.C., et al., Gram-scale synthesis of the p38alpha MAPK-inhibitor VX-745 for preclinical studies into Werner syndrome. Future Med Chem, 2010. 2(9): p. 1417-27.