NCB-0846

Epilepsy, one of the most common neurological disorders, is characterized by spontaneous recurrent seizures. Temporal lobe epilepsy (TLE) is one of the most common medically intractable seizure disorders. Traf2-and NcK-interacting kinase (TNIK) has recently attracted attention as a critical modulation target of many neurological and psychiatric disorders, but its role in epilepsy remains unclear. In this study, we hypothesized the involvement of TNIK in epilepsy and investigated TNIK expression in patients with intractable TLE and in a pilocarpine-induced rat model of epilepsy by western blotting, immunofluorescence, and immunohistochemistry. A pentylenetetrazole (PTZ)-induced epilepsy rat model was used to determine the effect of the TNIK inhibitor NCB-0846 on behavioral manifestations of epilepsy. Coimmunoprecipitation (Co-IP)/mass spectrometry (MS) was used to identify the potential mechanism. Through Co-IP, we detected and confirmed the main potential TNIK interactors. Subcellular fractionation was used to establish the effect of NCB-0846 on the expression of the main interactors in postsynaptic density (PSD) fractions. We found that TNIK was primarily located in neurons and decreased significantly in epilepsy model rats and TLE patients compared with controls. NCB-0846 delayed kindling progression and decreased seizure severity. Co-IP/MS identified 63 candidate TNIK interactors in rat hippocampi, notably CaMKII. Co-IP showed that TNIK might correlate with endogenous GRIA1, SYN2, PSD-95, CaMKIV, GABRG1, and GABRG2. In addition, the significant decrease in GRIA1 in hippocampal total lysate and PSDs after NCB-0846 treatment might help modify the progression of PTZ kindling. Our results suggest that TNIK contributes to epileptic pathology and is a potential antiepileptic drug target.

Hepatic fibrosis is driven by deposition of matrix proteins following liver injury. Hepatic stellate cells (HSCs) drive fibrogenesis, producing matrix proteins, including procollagen I, which matures into collagen I following secretion. Disrupting intracellular procollagen processing and trafficking causes endoplasmic reticulum stress and stress‐induced HSC apoptosis and thus is an attractive antifibrotic strategy. We designed an immunofluorescence‐based small interfering RNA (siRNA) screen to identify procollagen I trafficking regulators, hypothesizing that these proteins could serve as antifibrotic targets. A targeted siRNA screen was performed using immunofluorescence to detect changes in intracellular procollagen I. Tumor necrosis factor receptor associated factor 2 and noncatalytic region of tyrosine kinase‐interacting kinase (TNIK) was identified and interrogated in vitro and in vivo using the TNIK kinase inhibitor NCB‐0846 or RNA interference‐mediated knockdown. Our siRNA screen identified nine genes whose knockdown promoted procollagen I retention, including the serine/threonine kinase TNIK. Genetic deletion or pharmacologic inhibition of TNIK through the small molecule inhibitor NCB‐0846 disrupted procollagen I trafficking and secretion without impacting procollagen I expression. To investigate the role of TNIK in liver fibrogenesis, we analyzed human and murine livers, finding elevated TNIK expression in human cirrhotic livers and increased TNIK expression and kinase activity in both fibrotic mouse livers and activated primary human HSCs. Finally, we tested whether inhibition of TNIK kinase activity could limit fibrogenesis in vivo. Mice receiving NCB‐0846 displayed reduced CCl4‐induced fibrogenesis compared to CCl4 alone, although α‐smooth muscle actin levels were unaltered. Conclusions: Our siRNA screen effectively identified TNIK as a key kinase involved in procollagen I trafficking in vitro and hepatic fibrogenesis in vivo.