PRN694 shows extended target residence time on ITK and RLK, enabling durable attenuation of effector cells in vitro and in vivo. PRN694 is an irreversible, highly selective, and effective covalent interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) dual inhibitor (IC50s: 0.3 nM and 1.4 nM, respectively).

RLK:1.4 nM, BLK:125 nM, BTK:17 nM, JAK3:30 nM, BMX:17 nM, ITK:0.3 nM, TEC:3.3 nM

PRN694 obviously decreases NK cell FcR-induced killing at concentrations exceeding 0.37 μM. PRN694 inhibits TEC, BTK, BMX, BLK, JAK3 with IC50s of 3.3, 17, 17, 125, 30 nM, respectively. Immunoblot analysis of TCR activation pathways shows that PRN694 blocks activation or nuclear translocation of NFAT1, JunB, pIκBα, and pERK. Results display inhibition of Ca2+ signaling with PRN694 at all concentrations above 1 nM. Day 6 flow cytometry analysis reveals that PRN694 significantly inhibits the anti-CD3/CD28-induced proliferation of both CD4 and CD8 T-cells (p<0.01)[1].

RN694 treatment also causes obviously lower weights relative to the vehicle (p<0.05). Colitis studies show reduced numbers of CD4+ T cells present in the colonic epithelium of PRN694-treated mice compare with controls. The PRN694 occupancy of ITK is 98, 95, and 54% at 1, 6, and 14 h, respectively. The concentrations of PRN694 in the plasma are 2.8, 0.66, and 0.027 μM at 1, 6, and 14 h, respectively. At 14 h, the plasma level of PRN694 is over 10 fold lower than the IC50 in whole blood [1][2].