The signal-to-basal ratio (S/B) in this plate was 2 . 5-fold, with a CV of 11% and a Z factor of 0. 62. == Drug repurposing screen to identify active compounds that block Ebola VLP entry == We previously selected 600 FDA-approved drugs from our collection for a cancer project that eliminated certain compounds, including immunosuppressive drugs. of blocking Ebola VLP entry into cells. These 53 active compounds can be divided into categories including microtubule inhibitors, estrogen receptor modulators, antihistamines, antipsychotics, pump/channel Dovitinib Dilactic acid (TKI258 Dilactic acid) antagonists, and anticancer/antibiotics. Several of these compounds, including Dovitinib Dilactic acid (TKI258 Dilactic acid) microtubule inhibitors and estrogen receptor modulators, had previously been reported to be active in BSL-4 infectious Ebola virus replication assays and in pet model studies. Our assay represents a robust, effective and rapid high-throughput screen for the identification of lead compounds in drug development for the treatment of Ebola virus infection. Keywords: Antipsychotics, drug repurposing screen, Ebola virus, Ebola virus glycoprotein, estrogen receptor modulator, microtubule inhibitor, virus entry, VP40 == INTRODUCTION == The current outbreak of Ebola virus disease in West Africa has escalated to a scale not seen previously. As the outbreak continues, the export of virus infections to other regions in the world is occurring, accompanied in some cases by secondary infections. 1Currently, there is no proven effective treatment for Ebola virus infection. Although antibody-based therapy Tpo has been shown to be effective in a macaque model and has been used for the treatment of a few patients, the current supply of such drugs is very limited. 2The estimated mortality rate of the current Ebola outbreak is approximately 70%, which is dramatically high. 3Although a specific vaccine might eventually control Ebola virus infection, its development and deployment may take some time. 4, 5Therefore, drug repurposing screens that identify approved drugs with the potential for new indications might be a good approach to rapidly discover and develop anti-Ebola virus drugs for the treatment of patients with Ebola virus infection. We applied a previously developed Ebola virus-like particle (VLP) entry assay for a drug repurposing screen of Food and Drug Administration (FDA)-approved drugs. 6The Ebola VLP used in this assay is comprised of two proteins: glycoprotein (GP) and the matrix protein VP40 fused to a beta-lactamase reporter enzyme, which can be used to monitor VLP entry in a regular biosafety level 2 (BSL-2) cell model. We miniaturized this assay into a Dovitinib Dilactic acid (TKI258 Dilactic acid) 1536-well plate format for high-throughput screening (HTS). This initial drug repurposing screen led to the identification of 53 previously approved drugs with Ebola VLP entry-blocking activity. Although the activities of these drugs should be further confirmed using fully Dovitinib Dilactic acid (TKI258 Dilactic acid) infectious Ebola virusesin vitroandin vivoin BSL-4 laboratories, we herein publish all of our data for quick access by other researchers interested in further studies. Using the assay described, we plan to conduct more comprehensive compound screening to identify additional lead compounds for drug development to Dovitinib Dilactic acid (TKI258 Dilactic acid) treat Ebola virus infection. == MATERIALS AND METHODS == == Materials == Ebola VLPs containing a beta-lactamase-fused VP40 protein (EBOV BlaVP40) and GP were produced in Dr Garca-Sastre’s lab, as previously described. 6LiveBLAzer FRETB/G Loading Kit with CCF2-AM and Opti-MEM reduced serum medium were purchased from Life Technologies (Carlsbad, CA, USA). An adenosine triphosphate (ATP) content cell viability assay kit was purchased from Promega (Madison, WI, USA). Polystyrene plates (384-well and 1536-well black, clear bottom, sterile, tissue culture treated) were purchased from Greiner Bio-One (Monroe, NC, USA). A FDA-approved drug collection of 600 compounds was originally prepared at the National Center for Advancing Translational Sciences (NCATS) for a personalized cancer treatment project. This collection excludes certain drugs, such as those known to be immunosuppressive, topically applied drugs, and those for approved use in animals. In a follow-up screening, we used an NCATS-approved drug collection of 2816 compounds that was previously assembled. 7All of the compounds were dissolved as a 10 mM stock solution in dimethyl sulfoxide (DMSO) and diluted in DMSO at a 13 dilution to generate six concentrations in 384-well plates, followed by reformatting into three 1536-well compound source plates for HTS. == Cell culture methods == HeLa cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, GE healthcare,.