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Shigella flexneri is a facultative intracellular pathogen that causes shigellosis, a human diarrheal disease characterized by the destruction of the colonic epithelium. Novel antimicrobial compounds to treat infections are urgently needed due to the proliferation of bacterial antibiotic resistance and lack of new effective antimicrobials in the market. Our approach to find compounds that block the Shigella virulence pathway has three potential advantages: (i) resistance development should be minimized due to the lack of growth selection pressure, (ii) no resistance due to environmental antibiotic exposure should be developed since the virulence pathways are not activated outside of host infection, and (iii) the normal intestinal microbiota, which do not have the targeted virulence pathways, should be unharmed. We chose to utilize two phenotypic assays, inhibition of Shigella survival in macrophages and Shigella growth inhibition (minimum inhibitory concentration), to interrogate the 1.7 M compound screening collection subset of the GlaxoSmithKline drug discovery chemical library. A number of secondary assays on the hit compounds resulting from the primary screens were conducted, which, in combination with chemical, structural, and physical property analyses, narrowed the final hit list to 44 promising compounds for further drug discovery efforts. The rapid development of antibiotic resistance is a critical problem that has the potential of returning the world to a “pre-antibiotic” type of environment, where millions of people will die from previously treatable infections. One relatively newer approach to minimize the selection pressures for the development of resistance is to target virulence pathways. This is anticipated to eliminate any resistance selection pressure in environmental exposure to virulence-targeted antibiotics and will have the added benefit of not affecting the non-virulent microbiome. This paper describes the development and application of a simple, reproducible, and sensitive assay to interrogate an extensive chemical library in high-throughput screening format for activity against the survival of Shigella flexneri 2457T-nl in THP-1 macrophages. The ability to screen very large numbers of compounds in a reasonable time frame (~1.7 M compounds in ~8 months) distinguishes this assay as a powerful tool in further exploring new compounds with intracellular effect on S. flexneri or other pathogens with similar pathways of pathogenesis. The assay utilizes a luciferase reporter which is extremely rapid, simple, relatively inexpensive, and sensitive and possesses a broad linear range. The assay also utilized THP-1 cells that resemble primary monocytes and macrophages in morphology and differentiation properties. THP-1 cells have advantages over human primary monocytes or macrophages because they are highly plastic and their homogeneous genetic background minimizes the degree of variability in the cell phenotype (1). The intracellular and virulence-targeted selectivity of our methodology, determined via secondary screening, is an enormous advantage. Our main interest focuses on hits that are targeting virulence, and the most promising compounds with adequate physicochemical and drug metabolism and pharmacokinetic (DMPK) properties will be progressed to a suitable in vivo shigellosis model to evaluate the therapeutic potential of this approach. Additionally, compounds that act via a host-directed mechanism could be a promising source for further research given that it would allow a whole new, specific, and controlled approach to the treatment of diseases caused by some pathogenic bacteria.