Abstract PURPOSE: Regulation of endogenous gene expression using synthetic chemicals, so called “Transcription therapy” is a unique and promising approach. Although we have reported in vitro and in vivo anti-tumor, anti-metastatic and various anti-disease efficacies of hairpin Pyrrole-Imidazole polyamide (hPIP), PIP still needs to improve on selectivity of target DNA sequences and sensitivity to bind specific sequences. Hence, we synthesized PIPs targeting transcription-factor-binding-sites with several different structures and tested their efficacy, selectivity and sensitivity in vitro as well as in vivo model. METHODS: We have developed an automatic system for PIP synthesis by using peptide synthesizer of PSSM8 (Shimazu) and synthesized hPIPs or cyclic PIPs targeting either the MMP9 AP1 site or NFkB site to evaluate sensitivity and specificity. Each PIP, which has a different beta-alanine position, was tested for target DNA binding by surface plasmon resonance (SPR) using BiaCore system. We also test cell invasion and migration in vitro and metastatic capability in an in vivo xenograft model. RESULTS: Each hPIPs targeting AP1 or NFkB site showed significant reduction of MMP9 expression in MDA-MB-231 cells and HeLa cells and metastatic capacity in vivo. hPIP with crossed beta-alanine position showed the almost same dissociation constant as those with parallel beta-alanine position. Cyclic PIPs also showed similar dissociation constant as hPIPs and showed better sequence specificity with reasonable efficacy at least in vitro analysis. CONCLUSION: The result suggested that various PIPs with different target sites or architectures give distinct efficacy, selectivity and sensitivity in at least in vitro cancer cell culture system. Depending on pharmacokinetic properties of PIP the most suitable PIP can be selected for the future trial as seen in combinatorial chemistry technique. Citation Format: Hiroki Nagase, Nobuko Koshikawa, Takayoshi Watanabe. Automatic synthesis of efficient transcription inhibitors as anti-cancer agents designing sequence-specific DNA-binding molecules. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5679. doi:10.1158/1538-7445.AM2013-5679