Kirk E. Hevener1, PharmD, PhD; Emily Price1, BS; Julian Hurdle2, PhD; Dianqing Sun3, PhD
1)Â Â Â Â Presentation Format Requested:Â Oral or Poster (oral preferred)
2)Â Â Â Â Corresponding Author E-mail Address:Â firstname.lastname@example.org
3)Â Â Â Â Title:Â A Translational Medicine Approach to Drug Discovery:Â Repurposing Screening against a Novel and Selective Antibacterial Target in Clostridium difficile
4)Â Â Â Â Authors:Â Kirk E. Hevener1, PharmD, PhD; Emily Price1, BS; Julian Hurdle2, PhD; Dianqing Sun3, PhD
5)Â Â Â Â Institutional Affiliations:
1Idaho State University, College of Pharmacy, Department of Biomedical and Pharmaceutical Sciences, 1311 E. Central Drive, Meridian, IDÂ 83642
2Texas A & M Health Science Center, Institute of Biosciences and Technology, Center for Infectious and Inflammatory Diseases, 2121 W. Holcombe Blvd, Houston, TX 77030
3University of Hawaii at Hilo, College of Pharmacy, Department of Pharmaceutical Sciences, 200 W. Kawili Street, Hilo, HI 96720
6)Â Â Â Â Key words:Â Drug repurposing, Clostridium difficile, antibacterial, FabK, FAS-II
7)Â Â Â Â Objective:Â Hypervirulent strains of Clostridium difficile greatly contribute to the overall morbidity and mortality of C. difficile infection (CDI), resulting in an estimated 14,000 deaths per year and a national burden in excess of $3 billion annually.Â There is an urgent need for the development and rapid translation of novel and selective antibacterial agents for the treatment of CDI.Â The FabK enzyme, a rate-limiting enzyme in the bacterial fatty acid synthesis pathway (FAS-II), presents a novel and potentially selective antibacterial target as it is differentially expressed in C. difficile versus non-pathogenic gut flora.Â Our previous studies have confirmed the essentiality of this enzyme using genetic knock-out and knock-down approaches and have also demonstrated that the enzyme plays a significant role in C. difficile sporulation, which is known to contribute to the high recurrence rates observed in hypervirulent strains.Â The objective of this study was to apply a drug repurposing strategy by screening an approved drug library against the C. difficile FabK enzyme to identify inhibitory agents with potential for rapid clinical translation.
8)Â Â Â Â Setting:Â An academic biomedical research laboratory at a college of pharmacy, with one graduate student and one research technician.
9)Â Â Â Â Methods:The fabK gene from C. difficile (strain 630) was expressed and purified using a standard prokaryotic system.Â A fluorescence-based biochemical assay following NADH consumption was developed, optimized and used for compound library screening.Â The assay is run in 100mM Tris pH 8, 100 ÂµM NH4Cl and 0.01% Triton-X, withCdFabK enzyme at 800 nM, NADH (cofactor) at 25 ÂµM, Butenoyl-CoA (substrate) at 400 ÂµM and tested compounds at a final concentration of 25 ÂµM.Â Three compound libraries were experimentally screened, the NIH Clinical Collection, consisting of 731 approved drugs, and the NCI Diversity and Natural Products sets, consisting of 1700 compounds, combined.Â Compounds showing >50% inhibition of enzyme activity, in duplicate, were identified as hit compounds.
10)Â Results:Two hit compounds with CdFabK inhibitory activity were identified from the experimental screen of the NIH Clinical Collection, rabeprazole, a proton pump inhibitor, and mefenamic acid, a non-steroidal anti-inflammatory drug.Â Rabeprazole showed 68% inhibition of activity (average of duplicate trials) and mefenamic acid showed 73% activity inhibition, correlating to very promising low to sub-micromolar activity.Â Additionally, 65 compounds from the other compound libraries were identified as hits.
11)Â Interpretation:The hit compounds identified in these studies, while requiring further validation and characterization, have provided key structural and mechanistic insights into the inhibition of the CdFabK enzyme and may provide a framework for the discovery and rapid translation of novel therapeutic agents for CDI.