1. Small Molecules that Enhance the Pharmacological Effects of Oligonucleotides Melissa Porter 1, Bing Yang 1, Canhong Cao 1, Xin Ming 1, Emily Hull-Ryde 1, Joseph Maddry 2, Mark Suto 2, William Janzen 3 and Rudolph Juliano 1 1 Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 2 Southern Research Institute, Birmingham, AL 35255 3 Epizyme, Inc, 400 Technology Square, Cambridge, MA 02139 Center for Integrative Chemical Biology and Drug Discovery Funding: This work was supported by Federal Funds from the National Institute of Health, under grants #R01CA151964 and R21CA170332 to RLJ, and by an award from the University Cancer Research Fund of UNC. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Figures taken from: High Throughput Screening Identifies Small Molecules that Enhance the Pharmacological Effects of Oligonucleotides. Yang, Bing ; Ming, Xin; Cao, Canhong; Laing, Brian; Yuan, Ahu; Porter, Melissa; Hull-Ryde, Emily; Maddry, Joseph; Suto, Mark; Janzen, William; Juliano, Rudolph (in press Nucleic Acids Research) Acknowledgements Overview For almost two decades there has been strong interest in the potential use of antisense and siRNA oligonucleotides for cancer therapy. Despite much research, as well as FDA approval of the first antisense drug and the advent of multiple clinical trials in cancer and other diseases, the development of oligonucleotides as therapeutic agents has progressed only slowly. A major issue has been the poor delivery of these large, highly polar molecules to their sites of action in the cytosol or nucleus of target tissues and cells. We believe that this problem can be addressed using small molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. To this end, we designed and executed a high throughput screen of >150,000 compounds and identified three classes of oligonucleotide enhancing small molecules. These agents act on several forms of nucleic acids including antisense, splice switching and siRNA oligonucleotides, are active in the low micromolar range and can increase effectiveness of oligonucleotides to an extent comparable to cationic lipid transfection agents. Preliminary in vivo studies suggest that they are active when administered at non-toxic doses. Abstract Screen Results Dose Response and Cytotoxicity Conclusions Hi gh Throughput Screen Assay Primary author contact: maporter@unc.edu Goal: Use oligonucleotides (splice switching, antisense, SiRNA) as therapeutics Problem: Oligonucleotides are taken up by cells via endocytosis and get stuck in endosomes, limiting their activity Solution: Use small molecules to enhance delivery of oligonucleotides to their site of action by modulating their intracellular trafficking. Effect on ASO and SiRNA Oligo Subcellular Localization In Vivo Entrapment of oligonucleotides in endosomes is a major constraint on their pharmacological action Our studies demonstrate that HTS can identify small molecule enhancers of oligo activity and make the case for additional screening for more potent molecules with less toxicity. It is possible to release oligos from endosomes using a small molecule The intracellular trafficking, rather than the direct action of the oligo, is influenced by the small molecule Small molecules were screened based on their ability to greatly enhance luciferase induction via SSO623. We identified 3 compounds of interest. Hits were confirmed with dose response curves using the primary assay. False positives that increased spontaneous splice correction were identified and discarded. Confirmed hits were tested in the luciferase induction assay in a 24 well format and results normalized to cell protein concentration. Luciferase expression was enriched when the compounds were used in the 10-25  M range. To address whether the compounds directly affected the splicing process or the delivery of the oligos to the nucleus and cytosol, we looked at several types of oligos (SSO, ASO, siRNA), each having different mechanisms of action UNC7938 enhanced the ability of an siEGFP to reduce the levels of EGFP in a cell line stably expressing the reporter. Oligo accumulation was seen in the nucleus with compound treatment When EGFP654 transgenic mice were administered SSO with UNC7938, an increase in EGFP was observed in tissues HelaLuc705 cells contain a luciferase reporter interrupted by an abnormal intron. Reporter expression can be restored by a Splice Switching Oligo (SSO623). Cytotoxicity was assessed using Alamar Blue. Cells were exposed to compounds for 5 hours and then recovered an additional 24 hours. Only minimal toxicity was observed at a compound concentration of 10  M. UNC7938 enhanced the antisense knockdown of MDR1 in multi-drug resistant NIH-3T3 cells. The effect of UNC7938 was compared to a commercial cationic lipid, Lipofectamine 2000 (L2K). Treatment with UNC7938 led to a decrease of oligo in late endosomes (Rab7); presence in the lysosomes (LAMP1) was not affected