College of Pharmacy, Yeungnam University, Gyeongsan 38541, South Korea Discovery of Novel Small Molecule Inhibitors of TNF-α for Treating Inflammatory Bowel Disease: Ameliorating Effect of TI-1-172 on Rat Colitis Suhrid Banskota, Tara Man Kadayat, Pallavi Gurung, Eung-Seok Lee, Tae-Cheon Jeong, Jung-Ae Kim* College of Pharmacy, Yeungnam University, Gyeongsan 38541, South Korea Background/Aim: The important role of TNF-α in the pathogenesis of IBD has been proved by successful use of anti-TNF-α antibody therapeutics in the clinic. However, the systemic use of anti-TNF-α therapeutics elicits side effects such as serious infection, immunosuppression, and malignancies including lymphoma, due to suppression of normal functions of TNF-α which mediates inflammation against microorganism infection, regulates immune response, and induces apoptosis in certain cancer cells. Reminding that IBD is a disease of gut, the locally acting anti-TNF-α agents in the gut would be the best drug for treatment of IBD. In the present study, we searched and discovered small molecule inhibitor of TNF-α which is orally available and locally acting in the gut. Methods: A cell-based assay system was used: monocytes and colon epithelial cells (HT-29 cells) were co-cultured in the presence of TNF-α. Dual luciferase reporter assay system was used to study the inhibitory action of the selected chemicals in the TNF-α-induced transactivation of NF-κB and AP-1. The expression of adhesion molecules and cytokines were detected by Western blotting. Finally, the in vivo efficacy was evaluated in a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Results: Among 58 novel compounds of 2-benzylidene dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivative, TI-1-172 showed the strongest inhibitory effect on TNF-α-induced adhesion of monocytes to colon epithelial cells. Such inhibitory activity of the compounds correlated to its suppressive effect on expression of ICAM-1 and MCP-1, and NF-κB transcriptional activity. In addition, compound TI-1-172 significantly suppressed lipopolysaccharide (LPS)-induced expression of TNF-α gene, which corresponded to its additional inhibitory activity against AP-1 transcriptional activity, another transcription factor required for high level TNF-α expression. Oral administration of TI-1-172 ameliorated TNBS-induced rat colitis. Conclusion: Taken together, our in vitro and in vivo results demonstrate compound TI-1-172 as a locally acting small molecule anti-TNF-α drug candidate for IBD therapeutics development. Phenotype based screening of compounds Screening of compounds in vitro Pre-labeled U937 cells Pretreatment with compounds Treatment with TNF-α 1h 3h Monolayer of HT29 cells Wash 3 times with warm 1X PBS Pretreatment with compounds Measurement of Fluorescence with excitation at 485nm and emission at 520nm The IC50 values of selected compounds against TNF-α-induced adhesion of monocytes to colon epithelial cells [μM concentration except for 5-ASA (mM)] Compound a IC50c (μM) 1 5.75 ± 0.94 7 1.53 ± 0.23 25 0.95 ± 0.13 26 3.17 ± 0.21 32 3.72 ± 0.17 39 2.29 ± 0.19 41 0.83 ± 0.12 52 1.64 ± 0.29 54 1.68 ± 0.50 TI-1-172 0.50 ± 0.05 5-ASAb 20.4 ± 2.2 (mM) aμM concentration for compounds; bmM concentration of 5-ASA. cEach data represents mean ± S.E.M. from three different experiments performed in triplicate. Measurement of in vivo activity of compound Dissection of rat to isolate intestine Drug treatment for 5 days After 1 day Administration of TNBS intrarectally Explanations A B C Figure 2. TI-1-172 inhibits adhesion of monocyte to colon epithelial cells in a concentration-dependent manner. (A) Chemical structure of TI-1-172. HT-29 cells and 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescenin, Acetoxymethyl Ester (BCECF, AM) fluorescence-labeled U937cells were pretreated with different concentration of TI-1-172 for 1 h prior to the treatment with TNF-α (10 ng/mL). (B) Representative images of adhesion of monocyte to colon epithelial cell pictured by fluorescence microscopy. (C) Logarithm plot showing the % inhibition. Figure 1. Strategy for the design of compounds. A B C D Figure 3. Inhibition of adhesion molecule and cytokine expression by TI-1-172 is associated with the suppression of TNF-α-induced AP-1 and NF-κB activity. HT29 cells were pretreated with different concentration of TI-1-172 for 1 h prior to treatment with TNF-α (10 ng/mL) or LPS (1μg/mL) for 3 h and the extracted proteins were analyzed for (A) ICAM-1, MCP-1 and (B) TNF-α. *p < 0.05 compared to vehicle-treated control group. #p < 0.05 compared to TNF-α- or LPS-treated group. HT-29 cells were transfected with the luciferase reporter plasmid containing (C) human NF-ĸB and (D) human AP-1 responsive element. The reporter activity in the transfected cells was accessed after cells were pretreated with 5-ASA (20 mM) and indicated concentration od TI-1-172 prior to the treatment with TNF-α for 3 h. The bar graphs indicate mean ± S.E.M. from three independent experiments. *P < 0.05 compared to vehicle-treated control group. #P < 0.05 compared to TNF-α -treated group. A B C D Figure 4. Ameliorating effect of TI-1-172 on TNBS-induced colitis in rats. TNBS was rectally administered to rat to induce colitis. Control group received 50% ethanol in saline as a vehicle. (A) Record of body weight from day 0 to day 5. (B) The length and weight of colon after the dissection was measured. The bar graph represents the colon weight (g/cm). *P < 0.05 compared to vehicle-treated control group. #P < 0.05 compared to TNBS -treated group. (C) Macroscopic appearance of the removed tissue. (D) The amount of MPO was measured in the rat colon tissue using MPO assay kit. The result was expressed as mean ± S.E.M.