Kinase experiment:

ATP is dispensed into 384-well plates, chemical compounds (ISCK03: 2.5, 5, 10, 100 μM) are added by replicative plate, and recombinant human c-kit protein is added for the kinase reaction. Following a 45-min incubation at 37℃, the development reaction is carried out for 40 min at room temperature. After the reaction is stopped, the coumrain and fluorescein fluorescence-emission signals are detected[1].

Cell experiment:

To determine any cytotoxic effects of ISCK03 on 501mel cells, MTT assays are performed with various doses of ISCK03 (1, 5, 10 μM). 501mel cells are cultured with SCF alone (50 ng/mL) or SCF with ISCK03 for 48 h[1].

Animal experiment:

Mice: To induce the hair cycle, depilation of skin on the back of the female C57BL/6 mice is performed. Briefly, the hair is removed from anesthetized mice. The rat antimouse c-kit-neutralizing monoclonal antibody ACK2 is administered intraperitoneally (50 mg) every day. ISCK03 is administered orally once a day. On days 21–28, animals are sacrificed or analyzed for repigmentation of the newly regrown hair shaft. Skin is harvested and fixed in paraffin or frozen for immunohistochemical analyses[1].

ISCK03 is an inhibitor of SCF-mediated c-kit activation.

The signaling downstream of SCF/c-kit plays a key role in the development of various mammalian cells, such as mast cells, melanocytes, primordial germ cells, and hematopoietic progenitor cells.

In vitro: Previous study showed that the pretreatment of 501mel cells with ISCK03 could dose-dependently inhibit SCF-induced c-kit phosphorylation. ISCK03 could also inhibit the phosphorylation of p44/42 ERK mitogen-activated protein kinase that was known to be involved in SCF/c-kit downstream signaling. However, ISCK03 was not able to inhibit hepatocyte growth factor-induced phosphorylation of p44/42 ERK proteins [1].

In vivo: To determine the in vivo efficacy, ISCK03was orally administered to depilated C57BL/6 mice. Results showed that the oral administration of ISCK03 could induce the dose-dependent depigmentation of newly regrown hair, and this was reversed with cessation of ISCK03 treatment. In addition, the topical application of ISCK03 could promote the depigmentation of UV-induced hyperpigmented spots on n brownish guinea pig skin. Moreover, the Fontana–Masson staining analyses showed epidermal melanin was diminished in spots treated with ISCK03 [1].

Clinical trial: Up to now, ISCK03 is still in the preclinical development stage.

References:
[1] Y.  J. Na, H. S. Baek, S. M. Ahn, et al. [4-t-Butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03) inhibits SCF/c-kit signaling in 501mel human melanoma cells and abolishes melanin production in mice and brownish guinea pigs. Biochemical Pharmacology 74(5), 780-786 (2007).